COMPOSITIONS AND METHODS USING AMINOGLYCOSIDES TO BIND DNA AND RNA

I. CROSS-REFERENCE TO RELATED APPLICATIONS

1. This application claims benefit of U.S. Provisional Application No. 60/703,947, filed July 29, 2005, U.S. Provisional Application No. 60/704,147, filed July 29, 2005, U.S. Provisional Application No. 60/704,146, filed July 29, 2005, and U.S. Provisional Application No. 60/709,263, filed August 18, 2005, which are hereby incorporated herein by reference in their entirety.

IL ACKNOWLEDGEMENTS 2. The United States may have certain rights in the disclosed invention as it was at least in part funded by an NSF CAREER award (CHE/MCB-0134932).

III. BACKGROUND

3. Aminoglycoside antibiotics have been at the forefront of antimicrobial therapy for over half a century, (Waksman, S. A.;Lechevalier, H. A. J. Antibiot. 1949, 109, 305-309) and have garnered considerable attention in the past decade as the synthetic modifications to their structure have become more accessible. A number of groups have shown that many different RNA molecules can bind aminoglycosides: group I introns, a hammerhead ribozyme, the RRE transcriptional activator region from HIV (which contains the binding site for the Rev protein), the 5 '-untranslated region of thymidylate synthase rnRNA, and a variety of RNA aptamers from in vitro selection. (Wright, G. D.;Berghuis, A. M.;Mobashery, S. Adv. Exp. Med. Biol. 1998, 456, 27-69). It has been reported that aminoglycosides can stabilize DNA:RNA triplexes, hybrid duplexes, and that neomycin can even induce hybrid triplex formation (Arya, D. P.;Coffee, R. L., Jr.;Willis, B.; Abramovitch, A. I. J. Am. Chem. Soc. 2001, 123, 5385-5395, Xue, L.;Charles, I.;Arya, D. P. Chem. Commun. 2002, 70-71, Arya, D. P.;Coffee, R. L., Jr. Bioorg. Med. Chem. Lett. 2000, 10, 1897-1899). While it stabilizes DNA triplex structures, neomycin does not affect DNA duplex stability (under physiological ionic conditions) (Arya, D. P.;Coffee, R. L., Jr.;Willis, B.; Abramovitch, A. I. J. Am. Chem. Soc. 2001, 123, 5385-5395, Arya, D. P.;Coffee, R. L., Jr.;Charles, I. J. Am. Chem. Soc. 2001, 123, 11093-11094).

4. Small molecules that bind the DNA minor groove have been noticed for quite some time. The central responsibility for DNA affinity is the structural features of such small molecules; the crescent shape of the ligand matches the pitch of the DNA helix, snugly fitting within the minor groove to distribute various favorable contacts with the DNA bases. DNA

groove binding by small molecules is almost exclusively limited to the minor groove. Selective recognition of the major groove has remained elusive.

5. The structural features of the RNA minor groove are likely to be the reason for the shortage of ligands in this area of molecular recognition. The RNA duplex maintains an A- form conformation, which is characterized by a wide, shallow minor groove and a pinched, deep major groove. Therefore, the snug fit in the minor groove, otherwise accomplished with B-form DNA due to its characteristic narrow, deep minor groove, is absent. The known duplex RNA binders are more synthetically challenging due to their size and complex structure. A structural scaffold for groove recognition in duplex RNA is therefore lacking when compared to duplex DNA.

6. Neomycin, an aminoglycoside, has been shown to bind different RNA structures (Stage, T. K., et al. RNA 1995, 1, 95-101; Tok, J. B. H., et al. Biochemistry 1999, 38, 199-206; Hermann, T., et al. Biopolymers 1998, 48, 155-165; Chow, C. S., et al. Chem. Rev. 1997, 97, 1489-1514; Tor, Y., et al. Chem. Biol. 1998, 5, R277-R283). Aminoglycosides (in particular neomycin) can also bind to other A-form structures (Arya, D. P., et al. J. Am. Chem. Soc. 2003, 125, 10148-10149). Neomycin stabilizes poly(dA).2poly(dT) (Arya, D. P., et al. Bioorganic and Medicinal Chemistry Letters 2000, 10, 1897-1899), small triplexes (Arya, D. P., et al. J. Am. Chem. Soc. 2003, 125, 3733-3744), DNA.RNA hybrid duplexes, RNA triplex, and hybrid triple helices (Arya, D. P., et al. J. Am. Chem. Soc. 2001, 123, 11093-11094). Aminoglycosides most likely bind in the major groove of these structures (much like RNA, as the A-form nucleic acids have a narrower major groove) (Arya, D. P., et al. J. Am. Chem. Soc. 2003, 125, 10148-10149; Arya, D. P., et al. Bioorg. Med. Chem. Lett. 2000, 10, 1897-1899; Arya, D. P., et al. J. Am. Chem. Soc. 2001, 123, 11093-11094; Arya, D. P., et al. J. Am. Chem. Soc. 2001, 123, 5385- 5395; Arya, D. P., et al. J. Am. Chem. Soc. 2003, 125, 3733-3744; Arya, D. P. In Topics in Current Chemistry: DNA Binders; Chaires, J. B., Waring, M. J., Eds.; Springer Verlag: Heidelburg, 2005; Vol. 253, pp 149-178).

7. Various glycoconjugated polymers (Lee, Y. C. FASEB J. 1992, 6, 3193), dendrimers (Zanini, D., et al. J. Amer. Chem. Soc. 1997, 2088), calixarenes (Fujimoto, T., et al. J. Amer. Chem. Soc. 1997, 6676), and nanospheres (Yoshijumi, A., et al. Langmuir 1999, 15) have been developed as glycocluster models and biomedical materials. DNA has also been used as a conformationally rigid scaffold of such glycocluster models (Kazunori, M., et al. J. Amer. Chem. Soc. 2001, 123, 357-358). In contrast to abundant glycosylated protein and lipids in nature, few examples of glycosylated nucleic acids have been reported. A few reports discuss

glycosylated nucleosides (Lichtenstein, J., et al. Journal of Biological Chemistry 1960, 235, 1134-1141; Lehman, I. R., et al. Journal of Biological Chemistry 1960, 235, 3254-3259; Ehrlich, M., et al. Journal of Biological Chemistry 1981, 256, 9966-9972) and their role in gene expression (Borst, P., et al. Molecular and Biochemical Parasitology 1997, 90, 1-8; van Leeuwen, F., et al. Molecular and Biochemical Parasitology 2000, 109, 133-145). The presence of these glycosylated nucleosides modulates the functions of Escherichia coli bacteriophages of the T-even series (Kornberg, A., et al. DNA Replication; W. H. Freeman Co.: New York, 1992), genome of Trypanosoma brucei (a protozoan causing African sleeping sickness-as well as in phages) (Gommers-Ampt, J. H., et al. Cell 1993, 75, 1129-1136) and Diplonema (a small phagotrophic marine flagellate) (VanLeeuwen, F., et al. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 2366-2371). The glucose residues of modified-DNA reside within the major groove and render the DNA inaccessible by enzymes (van Leeuwen, F., et al. Anal. Biochem. 1998, 258, 223-229) and help the pathogen to escape degradation by host restriction enzymes. A limitation however in the synthesis of multivalent glycoconjugates has been the complexity of their synthesis and the difficulty in their purification (Dubber, M., et al. J. Bioconjug. Chem. 2003, 14, 239-246). Automated (solid phase) synthesis of such conjugates can allow their custom tailoring to the requirements of biological assays (Dubber, M., et al. J. Bioconjug. Chem. 2003, 14, 239-246). Needed are carbohydrates that can be conjugated to DNA without affecting the structure and stability of DNA, allowing the DNA to bind to their nucleic acid target (e.g., RNA) with specificity. Such conjugates would be of interest as a new class of artificial glycoconjugate materials applicable to cell-targeted gene therapy techniques.

8. Thus, compositions which are capable of binding B-form duplex DNA and derivatives are needed, as well as new compositions and mechanisms for binding A-form duplex RNA and derivatives and new compositions and mechanisms for binding single stranded DNA and RNA and derivatives.

IV. SUMMARY

9. Disclosed are methods and compositions related to aminoglycoside dimers and aminoglycoside conjugates, and their uses.

V. BRIEF DESCRIPTION OF THE DRAWINGS

10. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description illustrate the disclosed compositions and methods.

11. Figure 1 shows structures/ pKas of aminoglycosides with a central ribose.

^' "fit "Figufe ^sh ^' dws structures of aminoglycosides (kanamycin and gentamicin families).

13. Figure 3 shows base interactions in parallel (pyrimidine motif top) and antiparallel (purine motif bottom) triple helices.

14. Figure 4 shows variation of triplex melting (T _m 3→ 2) and duplex melting (T _m 2→ 1) of poly(dA)»2poly(dT) as a function of increasing neomycin concentration; r _< i _b ⁼ drug [neomycin]/base triplet ratio.

15. Figure 5 shows effect of aminoglycoside antibiotics on the melting of poly dA»2polydT triplex (r _db =l-67). Number of amines in each antibiotic is shown in parenthesis.

16. Figure 6 shows structures of some groove binders known to bind duplex DNA

17. Figure 7 shows the effect of 10 mM groove binders on the DNA triplex melt, poly(dA)«2poly(dT) (black bars) and the duplex melt, poly(dA)«poly(dT) (striped bars). Distamycin does not show T _m 3→ 2 transition (20°C). PEH Pentaethylene hexamine.

18. Figure 8a shows an ITC profile of 5 '-dA ₁₂ -x-dT ₁₂ -x-dT ₁₂ -3 ' (4 mM/strand) titrated with neomycin (500 mM) in 10 mM sodium cacodylate, 0.5 mM EDTA, 150 mM KCl, pH 6.8 at 2O ⁰ C. Figure 8b shows corrected injection heats plotted as a function of the [drug]/[DNA] ratio. The corrected injection heats were derived by integration of the ITC profile shown in Fig. 5a, followed by subtraction of the corresponding dilution heats derived from control titrations of drug into buffer alone. The data points reflect the experimental injection heats, while the solid line reflects calculated fit of the data.

19. Figure 9 shows charge/shape complementarity of neomycin to the triplex W-H groove: Electrostatic surface potential maps of neomycin approaching the W-H groove of the triplex (left), and neomycin buried in the triplex groove (right).

20. Figure 10 shows the effect of added aminoglycoside (ra _b ⁼ 0.66) on the stabilization of rA«dT duplex (gray) and on inducing rA»2dT triplex (black) .Number of amines in each aminoglycoside is shown in parenthesis.δT _m 3→ 2 is calculated by assuming a T _m 3→ 2 of 10°C in the absence of neomycin (no transition seen).

21. Figure 11 shows structures of neomycin, aminoacridines, and the neomycin- acridine conjugate.

22. Figure 12 shows competition dialysis results of neo-acridine (1 mM) with various nucleic acids; 180 mL of different nucleic acids (75 mM per monomelic unit of each polymer) were dialyzed with 400 mL of 1 mM neo-acridine in BPES buffer (6 mM Na ₂ HPO ₄ , 2 mM NaH ₂ PO ₄ , 1 mM Na ₂ EDTA, 185 mM NaCl, pH 7.0) solution for 72 h.

"23': Figure 1"3 'shows competition dialysis results (difference plots, with calf thymus DNA as reference) of 9-aminoacridine, quinacrine, and neo-acridine (1 mM) with various nucleic acids. Experimental conditions were identical to those for Fig. β.Maximum binding of neo-acridine is observed with nucleic acids that can adopt the A-type conformation.

24. Figure 14 shows competition dialysis results of 100 nM drug: difference plots, neo-acridine minus 9-aminoacridine (left) and neo-acridine minus quinacrine (right). 180 mL of different nucleic acids (7.5 mM per monomeric unit of each polymer) were dialyzed with 400 mL of 100 nM ligand in BPES buffer (6 mM Na2HPO4, 2 mM NaH2PO4, 1 mM Na2EDTA, 185 mM NaCl, pH 7.0) solution for at least 24 h.

25. Figure 15 shows conformations of an A-type duplex (left) and a B-type duplex (right), generally seen for RNA»RNA and DNA»DNA duplexes, respectively. The B-form duplex has a much wider major groove.

26. Figure 16 shows charge and shape complementarity of neomycin to the A-form major groove: Computer models of neomycin docked in the major groove of A-form DNA (left), and neomycin buried in the B-form major groove (right).

27. Figurel7 shows reagents and conditions: i a 5-trifluoroacetamido-l-pentanol, PPh ₃ , DIAD, dioxane, r.t, 2 h, 84%; i b HCl, EtOH, 0°C, quant.; ii a 2-(3,4-diaminophenyl)-6- (l-methyl-4- piperazinyl) benzimidazole, HOAc, reflux, 4 h, 38%; ii b K ₂ CO ₃ in 5:2 MeOH:H2O, r.t., overnight, 94%; Ui l,l'-thiocarbonyldi-2(lH)-pyridone, cat. DMAP, CH ₂ C12, r.t. 20 h, 95%; iv 3, pyridine, r.t., overnight, 72%; v 1:1 CH ₂ Cl ₂ , TFA, r.t., 3 h, quant.

28. Figure 18 shows UV melting profile of poly(dA)«2poly(dT) in the absence (a) and presence of 2 μM neomycin (b), 2 mM Hoechst 33258 (c), 2 mM Neomycin+2 mM Hoechst 33258 (d), and 2 mM Hoechst-neomycin conjugate (e). Samples of DNA (15 mM/base triplet) in buffer (10 mM Na cacodylate, 0.5 mM EDTA, 150 mM KCl, pH 7.20) containing ligand were analyzed for UV absorbance at 260 nm from 20-95°C using a temperature gradient of 0.2°C min ^"1 .

29. Figure 19a shows bar graph of δT _m for 22-mer duplexes in the presence of 4 mM Hoechst 33258 and 4 mM neomycin-Hoechst 33258 7 obtained from UV melting profiles (solution conditions were identical to those for Fig. 14). Figure 19b Computer model of neomycin-Hoechst 33258 docked in the DNA major-minor grooves.

30. Figure 20 shows structures of neomycin-DNA and kanamycin-DNA dimers Aminoglycoside-Nucleic Acid Interactions: The Case for Neomycin 173.

"ftf figure 2 ^" ϊ a sh'ows the structure of a generic aminoglycoside-DNA/PNA conjugate. Figure 21b shows synthesis of neomycin-DNA conjugate on the solid phase.

32. Figure 22 shows RNA Sequences at the 3'end of the 16sRNA as potential hybrid duplex targets.

33. Figure 23 shows the ribosomal 16s RNA sequence as potential ssRNA and dsRNA target.

34. Figure 24 shows ψ element of packaging region as a biologically significant RNA target.

35. Figure 25 shows secondary structure of the Rev-Response-Element (RRE).

36. Figure 26 shows secondary structure of the Trans-activating-region (TAR) element of HTV-I found to bind aminoglycosides.

37. Figure 27 shows structure of neomycin-neomycin and neomycin-tobramycin dimer used in the study.

38. Figure 28 shows UV melting profiles of Poly(dA)»2Poly(dT) in the presence of 150 mM KCl at the indicated drug concentrations. [DNA]=I 5 μM/base triplet. Solution conditions: 10 mM sodium cacodylate buffer, 0.5 mM EDTA, pH 7.2. Samples were heated from 20 to 95 °C at 5 deg/min, the annealing(95-20°C) and the melting (20-95 ⁰ C) were conducted at 0.2 deg/min, and the samples were brought back to 20°C at a rate of 5 deg/min.

39. Figure 29 shows a Job Plot of dA ₁₆ (1.25 μM/strand) and dT ₁₆ (1.25 μM/strand) in the presence of added neomycin (left, r _db =0.66) and neomycin-tobramycin dimer (right). Solution conditions: 10 mM sodium cacodylate, 0.5 mM EDTA, pH 6.8 at 10 ⁰ C.

40. Figure 30 shows (a) ITC profile of poly(dA).poly(dT) (60μM/base pairs) titrated with neomycin-tobramycin conjugate (200 μM); (b) Corrected injection heats plotted as a function of the [drug]/[ poly(dA).poly(dT)] ratio; (c) ITC profile of poly(dA)»poly(dT) (60μM/base pairs) titrated with neomycin-neomycin conjugate (200 μM); (d) Corrected injection heats plotted as a function of the [drug]/[ poly(dA).ρoly(dT)] ratio. Condition: 10 mM sodium cacodylate, 0.5 mM EDTA, 150 mM KCl, pH 6.8 at 2O ⁰ C.

41. Figure 31 shows scheme for synthesis of Hoechst-diamine (a) HO(CH ₂ ) ₂ O(CH ₂ ) ₂ O(CH ₂ ) ₂ OTs, PPh ₃ , DIAD, dioxane, 78%; (b) H ₂ N(CH ₂ ) ₃ O(CH ₂ ) ₃ O(CH ₂ ) ₃ NHCOCF ₃ , K ₂ CO ₃ , NaI, DMF, 61%; (c) (i) trifluoroacetic anhydride, pyridine, NEt ₃ , 58%; (d) (i) HCl(g), MeOH, quant, (ii) 4-[5-(4-Methyl-piperazin-l- yl)-lH-benzimidazol-2-yl]-benzene-l,2-diamine, MeOH, HOAc, 28%.

" ^" 42 ^{" '} Figure ϊ2 ^' "shows reaction of diamine 5 with neomycin isothiocyanate 6 (a) 5, pyridine, DMAP, 45%; (b) 7, TFA/CH ₂ C1 ₂ , trace ethanedithiol, 85%.

43. Figure 33 shows reagents prepared according to published procedures.

44. Figure 34 shows ITC profile of PolydA.2PolydT (30 μM/base) titrated by neomycin-tobramycin conjugate (200 μM) in 10 mM cacodylate, 0.5 mM EDTA, 150 mM KCl, pH 6.8 at 2O ⁰ C. 5 μl/inj; 300 sec/inj; 300 rpm stirring; 20 sec inj duration; 2 sec filter; 1.426 ml cell volume.

45. Figure 35 shows UV Melting of polyA»polyU. Profiles of RNA alone and in the presence of neomycin and NHl. [RNA] = 20 μM; [ligand] = 6 μM; Buffer: 10 mM PIPES, 1 mM EDTA, 100 mM NaCl, pH 7.0.

46. Figure 36 shows CD-detected binding of polyA«polyU and NHl . Small aliquots of concentrated ligand (500 μM) were added to a solution of RNA (40 μM) with stirring before scanning sample from 350 to 220 nm; Buffer 10 mM PIPES, 1 mM EDTA, 100 mM NaCl, pH 7.0. 47. Figure 37 shows CD detected melting of poly(A) 'PoIy(U) at 266 nm. [RNA] = 60 μM; [NHl] = 15 μM; Buffer: 10 mM PIPES, 1 mM EDTA, 100 mM NaCl ₅ pH 7.0.

48. Figure 38 shows CD detected melting of poly(A) "PoIy(U) + NHl monitored at 266 nm (A) and 342 nm (B). [RNA] = 60 μM; [NHl] = 15 μM; Buffer: 10 mM PIPES, 1 mM EDTA, 100 mM NaCl, pH 7.0. The CD at 266 nm represents RNA conformational changes, whereas 342 nm represents NHl (complexed with RNA) conformational changes.

49. Figure 39 shows fluorescence emission scans of NHl titration with polyA»polyU. A solution of NHl (333 nM) was titrated with a concentrated solution of RNA and mixed well before excitation at 342 nm. [RNA] ranged from 0.36 to 32 μM. Buffer: 10 mM PIPES, 1 mM EDTA, 100 mM NaCl, pH 7.0. 50. Figure 40 shows ITC of Neomycin and NHl binding to polyA»polyU. Figure 6A shows neomycin (10 μL injections of 150 μM) titrated into 40 μM RNA. Figure 6B shows NHl (10 μL injections of 100 μM) titrated into 40 μM RNA. Heat burst curves generated from binding were processed and curve fit using Origin 5.0. Buffer: 10 mM PIPES, 1 mM EDTA, 100 mM NaCl, pH 7.0. 51. Figure 41 shows viscometric analysis of poly(A)»poly(U) with various ligands.

RNA solutions (100 uM) were titrated with respective drug (500 μM) and corresponding flow times were recorded in triplicate with deviation less than 0.1 second. Buffer: 10 mM PIPES, 1 mM EDTA, 100 mM NaCl, pH 7.0.

^' 52: Figured shows UV Melting of r(CGCAAAUUUGCG) ₂ (SEQ ID NO:88). Profiles of RNA alone and in the presence of NHl. [RNA] = 2 μM duplex ^"1 ; [ligand] = 4 μM; Buffer: 10 mM PIPES, 1 mM EDTA, 100 mM NaCl, pH 7.0.

53. Figure 43 shows fluorescence titration of r(CGC AAAUUUGCG) ₂ (left) (SEQ ID NO:88) and r(CGCAAGCUUGCG) ₂ (right) (SEQ ID NO:89) into NH-I. A 2 mL solution of NHl (1 μM) was scanned after successive small aliquots of a concentrated RNA duplex solution (40 or 60 μM) and sufficient mixing. Excitation: 342 nm; Emission: 390-600 nm; slits: 4 nm; Buffer: PIPESlO (10 mM PIPES, 1 mM EDTA, 100 mM NaCl, pH 7.0); T = 25 ⁰ C.

54. Figure 44 shows hydrogen bonding interactions of Hoechst 33258 and d(CGCAAATTTGCG) ₂ (SEQ ID NO:90) extracted from pdb entry 296d. The bar lining the sequence (left) represents the binding site. Numbers over dashed lines represent the H-bond distances.

55. Figure 45 shows hydrogen bonding interactions of the Hoechst moiety of Hoechst 33258 and r(CGCAAAUUUGCG) ₂ (SEQ ID NO:88). The DNA coordinates were extracted from pdb entry Ial5. The bar lining the sequence (left) represents the binding site. Numbers over dashed lines represent the H-bond distances.

56. Figure 46 shows hydrogen bonding interactions of the Hoechst moiety of NHl and d(CGCAAATTTGCG) ₂ (SEQ ID NO:90). The DNA coordinates were extracted from pdb entry 296d. The bar lining the sequence (left) represents the binding site. Numbers over dashed lines represent the H-bond distances.

57. Figure 47 shows hydrogen bonding interactions of the Hoechst moiety of NHl and r(CGCAAAUUUGCG) ₂ (SEQ ID NO:88). The DNA coordinates were extracted from pdb entry Ial5. The bar lining the sequence (left) represents the binding site. Numbers over dashed lines represent the H-bond distances.

58. Figure 48 shows fluorescence titration of polyA»polyU and NHl. (left) Emission scans and (right) binding plot.

59. Figure 49 shows CD-detected binding of polyA»polyU and neomycin.

60. Figure 50 shows CD-detected binding of polyA»polyU and NHl.

61. Figure 51 shows ITC profile of Hoechst 33258 and polyA«polyU. Injections of ligand (40 x 5 μL of 100 μM) into 40 μM RNA were made at 2O ⁰ C. AU other conditions are identical to that reported in Methods.

62. Figure 52 shows scheme for synthesis of neomycin-DNA conjugate.

"$$ ^' ." figure 53 shows scheme for synthesis of neomycin isothiocyanate. Reaction conditions: (i) (a) (Boc) ₂ O, DMF, H ₂ O, Et ₃ N, 60 °C, 5 h, 60%; (b) 2,4,6- triisopropylbenzenesulfonyl chloride, pyridine, room temperature, 40 h, 50%; (c) H ₂ NCH ₂ CH ₂ SH, NaOEt/EtOH, room temperature, 18 h, 50%; (ii) l,l'-thiocarbonyldi- 2(lH)pyridone, 4-N,N-dimethylammopyridine and CH ₂ Cl ₂ (12 h, room temperature, 60%).

64. Figure 54 shows IR of neomycin isothiocyanate (recorded as a solution in CCl ₄ , and then manually subtracting the CCl ₄ peaks to get neomycin isothiocyanate 3 spectrum). Reaction conditions: (i) tetrachlorophthalimide, PPh ₃ , DIAD and THF (97%); (ii) ethylenediamine and THF; (iii) 4-methoxyphenyldiphenylmethyl (MmTr) chloride, triethylamine, 4-λ^N-dimethylammopyxidine and pyridine (60% for two steps); (iv) CNCH ₂ CH ₂ OP[N(zPr) ₂ ] ₂ , bis(diisoproρylammonium)tetrazolide and CH ₂ Cl ₂ (61%).

65. Figure 55 shows scheme for synthesis of deoxythymidine phophoramidite. Reaction conditions: (i) tetrachlorophthalimide, PPh ₃ , DIAD and THF (97%); (ii) ethylenediamine and THF; (iii) 4-methoxyphenyldiphenylmethyl (MmTr) chloride, triethylamine, 4-N, N-dimethylaminopyridine and pyridine (60% for two steps); (iv) CNCH ₂ CH ₂ OP[N(zPr) ₂ ] ₂ , bis(diisoρroρylammonium)tetrazolide and CH ₂ Cl ₂ (61%).

66. Figure 56 shows scheme for synthesis of dT16-neomycin conjugate. Reaction conditions: (a) (i) 6, lH-tetrazole and CH ₃ CN; (ii) capping of unreacted 5'-hydroxyl group; (iii) oxidation of P(IH) to P(V) with I ₂ , H ₂ O/pyridine/THF (iv) deprotection ofp- methoxyphenyldiphenylmethyl group from the 5'-amino group with 4% CCl ₃ CO ₂ H in CH ₂ Cl ₂ ; (b) (i) neomycin isothiocyanate 3, 4-N,N-dimethylaminopyridine and pyridine; (ii) β-elimination followed by deprotection from the solid support using cone. NH ₄ OH; (c) 1,4-dioxane solution containing 5% CF ₃ CO ₂ H and 1% m-cresol (v/v/v %).

67. Figure 57 shows HPLC profiles for dT ₁₆ -neomycin conjugate with Boc protected neomycin [9, plot (a)], dT ₁₆ -neomycin conjugate [10, plot (b)], and dT ₁₆ [plot (c)]; HPLC conditions for plot (a): buffer A: 100 mM of triethylammonium acetate in 10% of acetonitrile, pH=7.0; buffer A in 90% of acetonitrile; 10-50% of buffer B over buffer A during 20 min; flow rate was 1.5 ml/min; conditions for plot (b) and (c): buffer A: 25 mM of tris-HCl and 1 mM of EDTA, and ρH=8.0; buffer B: buffer A + IM of NaCl, and ρH=8.0; 30% of buffer B in buffer A for 2 min; 30-50% during 18 min; flow rate was 0.75 ml/min.

68. Figure 58 shows MALDI-TOF profiles for dT ₁₆ -neomycin conjugate with picolinic acid as a matrix.

69. Figure 59 shows synthesis of PNA T ₁₀ -Neomycin Conjugate

70. ^" Figure όϋ shows HPLC profile for T ₁₀ -Neo (12, Scheme 5) and Ti _O -LysNH ₂ ; Buffer Conditions for HPLC: Buffer A, 0.1% of trifluoroacetic acid in water; Buffer B, 0.08% of trifluoroacetic acid in acetonitrile: for PNA, 0-100% buffer B during 7 min; for PNA conjugate, 0-30% of buffer B over buffer A during 13 min; 30-100% buffer B during 2 min.

71. Figure 61 shows MALDI-TOF profile for PNA T ₁₀ -neomycin conjugate 12 (Scheme 5). α-cyano-4-hydroxycinnamic acid was used as a matrix.

72. Figure 62 shows Structure of the 7mer-neo conjugate.

73. Figure 63 shows scheme for synthesis of phosphoramidite 6. Reagents and Conditions: (i) (a) NeO-S-CH ₂ CH ₂ SCN, DMAP, pyridine (76%); (ii) (CF ₃ CO ₂ )O, Z-Pr ₂ EtN, CH ₂ Cl ₂ (75%); (iii) TBAF, DMF; and then DMTrCl, Pyridine, DMAP (78%); (iv) NCCH ₂ CH ₂ OP[N(zPr) ₂ ] ₂ , bis(diisoρropylammonium) tetrazolide and CH ₂ Cl ₂ (72%).

74. Figure 64 shows scheme for covalent attachment of neomycin to an oligonucleotide, (i) Deprotection with 4% trichloroacetic acid in CH ₂ Cl ₂ , and coupling with 3 in the presence of lH ^" -tetrazole followed by capping with acetic anhydride isn pyridine/THF solution, and oxidation with I ₂ in THF/H ₂ O/pyridine solution ; (ii) β-elimination followed by deprotection from the solid support using cone. NH ₄ OH; (c) 1,4-dioxane solution containing 3% CF ₃ CO ₂ H and 1% m-cresol (v/v/v %).

75. Figure 65 shows purification of deprotected conjugate by preparative anion exchange HPLC. Conditions: Buffer A: 25 mM tris-HCl and 1 mM EDTA, and pH=8.0; buffer B: buffer A + IM NaClO ₄ , and pH=8.0; 2% buffer B in buffer A for 2 min; 2-40% during 15 min; flow rate was 0.75 ml/min. X corresponds to deoxyuridine-neomycin conjugate base.

76. Figure 66 shows MALDI-TOF profile of 7mer-neomycin conjugate recorded with picolinic acid as the matrix and ammonium tartarate as the co-matrix.

77. Figure 67 shows conditions: Buffer A: 100 mM triethylammonium acetate and 5% of acetonitrile; buffer B: 95% of acetonitrile and 5% of 100 mM triethylammonium acetate, and pH=7.0; 1% -10% of buffer B over buffer A during 15 min, flow rate was 0.5 ml/min ; 10- 23% during 8 min, flow rate was 1.0 ml/min.

78. Figure 68 shows UV melting profiles of (a) rR:dY hybrid duplex in the absence (filled squares) and presence of 4 μM of neomycin (open circles); (b) rR:N-dY hybrid duplex in the absence of neomycin; (c) rR ^! :N-dY hybrid duplex in the absence of neomycin. The spectra were recorded at 260 nm in the presence of 60 mM Na ⁺ ion concentration. [DNA]=4 μM / strand. Buffer conditions: 10 mM sodium cacodylate, 0.1 mM EDTA, pH 7.0. The melting rate was 0.2°C/min.

79 ^' . Figure ^' W shows CD titration of 1 OμM of rR with dY (filled circle) and N-dY (open square) at 10 ⁰ C and pH 7.0 in cacodylate buffer. Buffer conditions: 10 mM sodium cacodylate, 0.5 mM EDTA, 60 mM total Na ⁺ , and pH 7.0.

80. Figure 70 shows ITC profile of rR single strand titrated against (a) dY single strand (b) neomycin conjugate (N-dY) at 10°C and pH 7.0 in sodium cacodylate buffer. Buffer conditions: 10 mM sodium cacodylate, 0.1 mM EDTA, pH 7.0 and 6OmM total Na ⁺ ions. Each heat burst curve is a result of a lOμl injection of 200μM of rR into 15μM of N-dY solution. A plot of corrected ITC injection heats as a function of the [rR]/[N-dY] ratio (rd _up ). The heats were derived by subtraction of the heats obtained by the titration of rR SS against the N-dY SS with the heats obtained by blank titration of the corresponding buffer vs. buffer. The data points reflect the experimental injection heats, while the line denotes calculated fit using a model for one set of binding.

VL DETAILED DESCRIPTION

81. Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

82. U.S. Provisional Application No. 60/703,947, entitled "AMINOGLYCOSIDE DIMERS AND THEIR USES" by Dev Priya Arya and Liang Xue, filed July 29, 2005; U.S. Provisional Application No. 60/704,147, entitled "CONJUGATES OF AMINOGLYCOSIDE ' AND MINOR GROOVE BINDERS AND THEIR USES" by Dev Priya Arya and Bert Willis, filed July 29, 2005; and U.S. Provisional Application No. 60/704,146, entitled "SINGLE- STRANDED RNA-BINDING AMINOGLYCOSIDE AND THEIR USES" by Dev Priya Arya and Ixudayasamy Charles, filed July 29, 2005 are hereby incorporated by reference in their entireties.

A. Definitions

83. As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

"^H. Rautϊ ^* ges"'can Be' expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that when a value is disclosed that "less than or equal to" the value, "greater than or equal to the value" and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value "10" is disclosed the "less than or equal to 10"as well as "greater than or equal to 10" is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point "10" and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

85. As used throughout, by a "subject" is meant an individual. Thus, the "subject" can include, for example, domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.) mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal. The subject can be a mammal such as a primate or a human.

86. "Treating" or "treatment" does not mean a complete cure. It means that the symptoms of the underlying disease are reduced, and/or that one or more of the underlying cellular, physiological, or biochemical causes or mechanisms causing the symptoms are reduced. It is understood that reduced, as used in this context, means relative to the state of the disease, including the molecular state of the disease, not just the physiological state of the disease.

87. By "reduce" or other forms of reduce means lowering of an event or characteristic. It is understood that this is typically in relation to some standard or expected

value, in other words ^' ϊtϊs ^" relative, but that it is not always necessary for the standard or relative value to be referred to. For example, "reduces phosphorylation" means lowering the amount of phosphorylation that takes place relative to a standard or a control.

88. By "inhibit" or other forms of inhibit means to hinder or restrain a particular characteristic. It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, "inhibits phosphorylation" means hindering or restraining the amount of phosphorylation that takes place relative to a standard or a control. ,

89. By "prevent" or other forms of prevent means to stop a particular characteristic or condition. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce or inhibit. As used herein, something could be reduced but not inhibited or prevented, but something that is reduced could also be inhibited or prevented. It is understood that where reduce, inhibit or prevent are used, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed. Thus, if inhibits phosphorylation is disclosed, then reduces and prevents phosphorylation are also disclosed.

90. The term "therapeutically effective" means that the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination. The term "carrier" means a compound, composition, substance, or structure that, when in combination with a compound or composition, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or composition for its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

91. Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises," means "including but not limited to," and is not intended to exclude, for example, other additives, components, integers or steps.

92. The term "cell" as used herein also refers to individual cells, cell lines, or cultures derived from such cells. A "culture" refers to a composition comprising isolated cells of the same or a different type.

93. The term "pro-drug" is intended to encompass compounds which, under physiologic conditions, are converted into therapeutically active agents. A common method for making a prodrug is to include selected moieties which are hydrolyzed under physiologic

23

conditions to reveal the desired molecule. In other embodiments, the prodrug is converted by an enzymatic activity of the host animal.

94. The term "metabolite" refers to active derivatives produced upon introduction of a compound into a biological milieu, such as a patient.

95. When used with respect to pharmaceutical compositions, the term "stable" is generally understood in the art as meaning less than a certain amount, usually 10%, loss of the active ingredient under specified storage conditions for a stated period of time. The time required for a composition to be considered stable is relative to the use of each product and is dictated by the commercial practicalities of producing the product, holding it for quality control and inspection, shipping it to a wholesaler or direct to a customer where it is held again in storage before its eventual use. Including a safety factor of a few months time, the minimum product life for pharmaceuticals is usually one year, and preferably more than 18 months. As used herein, the term "stable" references these market realities and the ability to store and transport the product at readily attainable environmental conditions such as refrigerated conditions, 2°C to 8°C.

96. References in the specification and concluding claims to parts by weight, of a particular element or component in a composition or article, denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

97. A weight percent of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.

98. Li this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:

99. "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

100. "Primers" are a subset of probes which are capable of supporting some type of enzymatic manipulation and which can hybridize with a target nucleic acid such that the enzymatic manipulation can occur. A primer can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art which do not interfere with the enzymatic manipulation.

it)f . Probes" are molecules capable of interacting with a target nucleic acid, typically in a sequence specific manner, for example through hybridization. The hybridization of nucleic acids is well understood in the art and discussed herein. Typically a probe can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art.

102. Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

103. Disclosed are the components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular aminoglycoside dimer is disclosed and discussed and a number of modifications that can be made to a number of molecules including the aminoglycoside are discussed, specifically contemplated is each and every combination and permutation of aminoglycoside and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C- D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.

B. Compositions

104. Several classes of compositions for targeting and binding nucleic acids are disclosed herein. Thus, provided herein is a method of binding a nucleic acid comprising administering any of the herein provided compositions. For example, one class are B-form

bihdersl ^" Tne ^' B-'fofm Birϊ&€rirβind B firorm duplex nucleic acid in the major groove and have a preference for binding AT rich regions. An example of this type of molecule would be an aminoglycoside dimer as disclosed herein. Another class of compositions for binding nucleic acids are A-form binders which bind A-form duplex nucleic acid and also prefer AT regions. An example of an A-form binder would be an aminoglycoside conjugated to a B-form minor groove binder, such as neomycin conjugated to Hoechst 33258. A third class of nucleic acid binders are single stranded nucleic acid binders, suich as single stranded RNA or DNA binders. These molecules typically bind single stranded nucleic acid with improved binding efficiency and specificity than the complementary strand would alone. An example of a single-stranded binder would be an aminoglycoside conjugated to a complementary nucleic acid strand, at either the 5' or 3' ends or internally, such as neomycin.

105. Also disclosed are combinations of each of these classes of binders, such as a single stranded RNA binder, with an aminoglycoside conjugated internally on a complementary nucleic acid, which is also conjugated on either the 5' or 3' end to an A-form binder, such as neomycin- Hoechst 33258. Any combination can be made, but specifically disclosed are A-form binders conjugated to single stranded binders, or B-form binder conjugated to a B-form major groove binder, for example.

106. In an effort to improve aminoglycoside-nucleic acid binding and develop more effective antibiotics, several novel neomycin conjugates have been reported, that include intercalator linked (Xue, L.;Charles, I.;Arya, D. P. Chem. Commun. 2002, 70-71, Arya, D. P.;Xue, L.; Tennant, P. J. Am. Chem. Soc. 2003, 125, 8070-8071) as well as dimeric aminoglycosides linked via a long chain-alkyl linkage. (Michael, K.;Wang, H.;Tor, Y. Bioorg. Med. Chem. 1999, 7, 1361-1371, Michael, K.;Tor, Y. Chem. Eur. J. 1998, 4, 2091-2098). The dimeric aminoglycosides were shown to be more effective RNA binders than the individual aminoglycosides (Michael, K.;Wang, H.;Tor, Y. Bioorg. Med. Chem. 1999, 7, 1361-1371). Disclosed herein, the remarkable charge and shape complementarity of neomycin to the larger W-H groove is shown. Several intercalator-neomycin conjugates have shown synergistic stabilization of the triplex structures as well. (Xue, L.;Charles, I.;Arya, D. P. Chem. Commun. 2002, 70-71). The presence of additional positive charges and the size of dimeric conjugates (FIG 22) relates to their role in enhanced triplex groove recognition (perhaps through simultaneous major-minor groove interactions). Disclosed herein, are nucleic acid stabilization results observed in the presence of a tobramycin-neomycin and neomycin-neomycin dimer (FIG

22), as well as other dϊm'efs Having the appropriate charge/shape complementartity as disclosed herein.

1. B-form binders -Such as the major groove of duplex DNA

107. A B-form binder is a composition which can bind B-form duplex nucleic acid. It has been shown herein that a dimer of an aminoglycoside, either homo or hetero dimer, can bind B-form duplex nucleic acid in a specific way, targeting AT rich regions, and that these dimers bind more tightly than the monomer alone. Provided are dimers of aminoglycosides which can be used to bind the major groove of duplex DNA as described herein. It is understood that these dimers can be made as described herein, and can have, for example a linker attaching them.

108. Thus, provided herein is a composition comprising a B-form dimer, wherein the B-form dimer comprises an aminoglycoside dimer. Thus, the B-form binders can comprise a dimer, two aminogloycosides, which can be attached via a linker. Furthermore, the dimers can be attached to other things, such as b-form major groove binders, such as a triplex strand of nucleic acid, or to B-form minor groove binders, such as Hoechst 33258.

109. Thus, provided herein is a composition comprising a dimer of a first aminoglycoside and a second aminoglycoside. Also provided is a composition comprising an aminoglycoside linked to an aminoalcohol or a polyamine.

110. Aminoglycosides can be associated or linked in any suitable manner. For example, aminoglycosides can interact or be linked non-covalently, ionically, or covalently. Non-covalent interactions can be or any type or combination of types. Thus, for example, aminoglycosides can interact through polar interactions, charge interactions, van der Waals forces, hydrophobic interactions, or any combination of these. Aminoglycosides can be covalently coupled in any suitable manner, either directly, via a linkage group, or via a linker. In a given delivery composition, different aminoglycosides can interact or be linked with each other in different ways.

111. Many coupling chemistries are known and can be adapted for use in coupling or linking aminoglycosides to other compositions, such as there herein disclosed minor groove binders, nucleic acids and other aminoglycosides. For example, crosslinking of aminoglycosides can be based on click chemistry. The term "click chemistry" refers to any crosslinking chemistry that is highly favorable under mild conditions and was first coined by Valerie Fokin and K. Barry Sharpless in regards to the triazole-forming reaction between an azide and an alkyne in aqueous environment (Rostovtsev et al., Angew. Chem. Int. Ed. 2002, 41, 2596-9). This crosslinking chemistry, which has been used in drug discovery (Lee et al, J. Am. Chem. Soc. 2003, 125,

9588-9; Lewis et άl.;Angew7Chem. Int. Ed. 2002, 41, 1053-7; Lewis et al, J. Am. Chem. Soc. 2004, 126, 9152-3), fluorogenic probes (Zhou and Fahrni, J. Am. Chem. Soc. 2004, 126, 8862-3), and cell surface engineering (Link et al, J. Am. Chem. Soc. 2004, 126, 10598-602; Agard et al, J. Am. Chem. Soc. 2004, 126, 15046-7), can involve the use of copperQ) as a catalyst (Arciello et al, Biochem. Biophys. Res. Commun. 2005, 327, 454-9; Smet et al, Hum. Exp. Toxicol. 2003, 22, 89-93; Seth et al, Toxicol In Vitro 2004, 18, 501-9) or can involve the use of catalyst-free click chemistry, which can be accomplished using, for example, electron-deficient alkynes (Li et al, Tetrahedron Lett. 2004, 45, 3143-3146). AU of the references disclosed in this paragraph are hereby incorporated by reference at least for their teaching of click chemistry.

112. In some aspects disclosed herein provided compositions, the first aminoglycoside and second aminoglycoside, aminoalcohol or polyamine are connected by a linker. A linker can be any chain, structure, or region (other than the aminoglycoside) that links aminoglycosides. The linker can have a branched linker structure. Any core or branched structure can form the junction of aminoglycosides. The linker can be anything allowing the connection of the two molecules such that binding takes place. For example, the linker can be a triazole formed by click chemistry. Linkers can also be carbon chains that can have oxygen, nitrogen or sulfer in between in any number and at any position, and can very from 0 to 20. Examples of linkers that can be used include (CH ₂ ) ₂ O(CH ₂ ) ₃ O(CH ₂ ) ₂ (CH ₂ ) ₃ O(CH ₂ ) ₂ O(CH ₂ ) ₃ ; (CH ₂ ) ₂ [O(CH ₂ ) ₂ ] ₂ O(CH ₂ ) ₂

; (CH ₂ ) ₃ O(CH ₂ ) ₄ O(CH ₂ ) ₃ ; (CH ₂ ) ₃ [O(CH ₂ ) ₂ ] ₂ O(CH ₂ ) ₃ ; (CH ₂ ) ₂ [O(CH ₂ ) ₂ ] ₃ O(CH ₂ ) ₂ ; (CH ₂ ) ₁₀ ; and (CH ₂ ) ₁₂ .

113. The herein provided compositions can bind double stranded DNA at a major groove binding site. The major groove binding site can be an AT rich region. Thus, the binding site can comprise at least 5 contiguous bases of adenosine. As an example, the binding site can comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous bases of adenosine. Thus, the binding site can comprise at least 5 contiguous bases of thymidine. As an example, the binding site can comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous bases of thymidine. Thus, the binding site can comprise at least 5 contiguous bases of adenosine or thymidine. As an example, the binding site can comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous bases of adenosine or thymidine.

114. The herein provided compositions can bind a 16 base polyA-polyT duplex with a Kd of less than or equal to 1 x 10 ^"6 , 1 x 10 ^"7 , 1 x 10 ^"8 , or 1 x 10 ^"9 .

115. The first and second aminoglycosides can comprise any aminoglycosides known in the art or provided herein. Thus, in some aspects of the herein provided compositions, the first

ammogrycόsfde comprises neomycin. In some aspects of the herein provided compositions, the second aminoglycoside comprises a neomycin. In some aspects, the first aminoglycoside comprises a tobramycin. In some aspects, the second aminoglycoside comprises a tobramycin.

116. The herein provided compositions can further comprise a major groove binder. The major groove binder can be any major groove binder known in the art or disclosed herein.

117. The herein provided compositions can further comprise a minor groove binder. The minor groove binder can comprise any minor groove binder known in the art or provided herein. Thus, the minor groove binder can comprise Hoechst 33258 or a polyamide, for example.

118. The herein provided compositions can further comprise an oligonucleotide. The herein provided compositions can further comprise a nucleic acid, wherein the sequence of the nucleic acid is capable of interacting with the major groove The oligonucleotide can be a nucleic acid, polynucleotide, or oligonucleotide known in the art or disclosed herein. In some aspects, the oligonucleotide can comprise a nucleic acid capable of forming a triplex nucleic acid within 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 bases of the dimer binding site.

119. The herein provided aminoglycoside dimers can be conjugated to a triplex- forming nucleic acid at the 5' end of the nucleic acid or 3' end of the nucleic acid. Alternatively, the herein provided aminoglycoside dimers can be covalently attached to a nucleotide and incorporated at any site within the triplex-forming nucleic acid. As an example, an aminoglycoside of the herein provided compositions can be covalently attached to the C5- position of 2'-deoxyuridine. The amino groups on rings I, It and IV (neomycin, Figure 1) are necessary in stabilizing and recognizing various nucleic acid forms. Thus, in some aspects, the nucleic acids are covalently attached to the aminoglycoside at the 5"-OH on the ribose of ring DI. A method for covalently attaching neomycin to the C5-position of 2'-deoxyuridine is provided herein (Example 6).

120. Also provided herein is a method of interacting with the major groove of a B duplex DNA molecule comprising incubating an aminoglycoside dimer provided herein with a B duplex DNA molecule.

121. Also provided herein is a method of inhibiting a protein from interacting with a double stranded DNA molecule comprising incubating an aminoglycoside dimer provided herein with the double stranded DNA.

2. A-form binders — Duplex RNA

122. Disclosed are A-form binders. An A-form binder is a composition capable of binding A-form duplex nucleic acid. An example of an A-form binder is NHl, as shown in the

examples. Disclosed ^' are aminoglycoside conjugates, which can be used to bind double stranded RNA as described herein. It is understood that these conjugates can be made as described herein, and can have, for example a linker attaching them. •

123. Provided herein is a composition comprising an A-form dimer, wherein the A-

> form dimer comprises an aminoglycoside conjugated to a minor groove binder. Thus, provided herein is a composition comprising a conjugate of a first aminoglycoside and a second minor groove binder. In some aspects of the herein provided compositions, the first aminoglycoside and second minor groove binder are connected by a linker. The linker can be any molecule capable of connecting the two molecules, such that the connected molecules can function. Carbon chains

3 that can have oxygen nitrogen or sulfer in between in any number and at any position, and can very from 0 to 20. For example, the linker can be glycol or alkyl in nature. For example, the linker can be glycol or alkyl in nature. Examples of linkers that can be used include (CH ₂ ) ₂ O(CH ₂ ) ₃ O(CH ₂ ) ₂ (CH ₂ ) ₃ .O(CH ₂ ) ₂ O(CH ₂ ) ₃ ; (CH ₂ ) ₂ [O(CH ₂ ) ₂ ] ₂ O(CH ₂ ) ₂ ; (CH ₂ ) ₃ O(CH ₂ ) ₄ O(CH ₂ ) ₃ ; (CH ₂ ) ₃ [O(CH ₂ ) ₂ ] ₂ O(CH ₂ ) ₃ ; (CH2) ₂ [O(CH ₂ ) ₂ ] ₃ O(CH ₂ ) ₂ ; (CH ₂ ) ₁₀ ; and

5 (CH ₂ )I ₂ .

124. The herein provided compositions can bind double stranded RNA at a major (aminoglycoside) and minor groove binding sites. Minor groove binders can preferentially bind AU-rich regions or GC-rich regions. Hoechst 33258 preferentially binds AU-rich regions of RNA, while polyamides can bind GC-rich regions. Thus, the major groove binding site can be an

0 AU rich region. Thus, the binding site can comprise at least 5 contiguous bases of adenosine. As an example, the binding site can comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous bases of adenosine. Thus, the binding site can comprise at least 5 contiguous bases of uridine. As an example, the binding site can comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous bases of uridine. Thus, the binding site can comprise at least

5 5 contiguous bases of adenosine or uridine. As an example, the binding site can comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous bases of adenosine or uridine. The major groove binding site can also be an GC rich region. The minor groove binder can be, for example, a polyamide.

125. The herein provided compositions can bind a 16 base polyA-polyU duplex with a

0 Kd of less than or equal to 1 x 10" ⁶ , 1 x 10" ⁷ , 1 x 10" ⁸ , or 1 x 10" ⁹ .

126. The herein provided compositions can also bind double stranded DNA at a major (aminoglycoside) and minor groove binding sites. The minor groove binder can be, for example, a polyamide. Thus, the major groove binding site can be an GC rich region.

127. The aminoglycoside of the provided composition can be any aminoglycoside known in the art or provided herein. Thus, in some aspects of the herein provided compositions, the aminoglycoside comprises neomycin, hi some aspects, the aminoglycoside comprises a tobramycin.

128. The minor groove binder can comprise any minor groove binder known in the art or provided herein. Thus, the minor groove binder can comprise Hoechst 33258 or polyamide.

In some aspects, the nucleic acid can comprise a nucleic acid capable of forming a triplex nucleic acid within 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 bases of the dimer binding site.

129. The amino groups on rings I, II and IV (neomycin, Figure 1) are necessary in stabilizing and recognizing various nucleic acid forms. Thus, in some aspects, the minor groove binders are covalently attached to the aminoglycoside at the 5-OH on the ribose of ring EL

130. The herein provided aminoglycoside conjugates can be conjugated to a triplex- forming nucleic acid at the 5' end of the nucleic acid or 3' end of the nucleic acid. Alternatively, the herein provided aminoglycoside conjugates can be covalently attached to a nucleotide and incorporated at any site within the triplex-forming nucleic acid. As an example, an aminoglycoside of the herein provided compositions can be covalently attached to the C5- position of 2'-deoxyuridine. A method for covalently attaching neomycin to the C5-position of 2'-deoxyuridine is provided herein (Example 6).

131. Also provided herein is a method of interacting with the maj or groove of a double-stranded RNA molecule comprising incubating an aminoglycoside conjugate provided herein with a double-stranded RNA molecule.

132. Also provided herein is a method of inhibiting a protein from interacting with a double-stranded RNA molecule comprising incubating an aminoglycoside conjugate provided herein with the double stranded DNA.

3. Single-stranded nucleic acid binders

133. A single stranded nucleic acid binder is a composition capable of binding a single stranded nucleic acid. An example of a single stranded nucleic acid binder is an oligonucleotide which is complementary to a target single strand sequence wherein the oligonucleotide is conjugated to an aminoglycoside. Disclosed are aminoglycoside conjugates, which can be used to bind single stranded nucleic acids, such as RNA as described herein. It is understood that these conjugates can be made as described herein, and can have, for example a linker attaching them.

1347 Targeting single stranded DNA: In some viruses DNA appears in a non-helical, single-stranded form. Because many of the DNA repair mechanisms of cells work only on paired bases, viruses that carry single-stranded DNA genomes mutate more frequently than they would otherwise. As a result, such species may adapt more rapidly to avoid extinction. The result would not be so favorable in more complicated and more slowly replicating organisms, however, which may explain why only viruses carry single-stranded DNA. These viruses presumably also benefit from the lower cost of replicating one strand versus two. If an ODN is coupled to Aminoglycoside dimer, the resulting duplex will be stabilized considerably because of the dimer binding to duplex target that forms.

135. Provided herein is a composition comprising a single-stranded nucleic acid binder, wherein the single-stranded nucleic acid binder comprises an oligonucleotide conjugated to an aminoglycoside. Thus, provided herein is a composition comprising a conjugate of an aminoglycoside and a nucleic acid. In some aspects of the herein provided compositions, the aminoglycoside and nucleic acid are connected by a linker. The linker can be glycol or alkyl in nature. Examples of linkers that can be used include

(CH ₂ ) ₂ O(CH ₂ ) ₃ O(CH ₂ ) ₂ (CH ₂ ) ₃ O(CH ₂ ) ₂ O(CH ₂ ) ₃ ; (CH ₂ ) ₂ [O(CH ₂ ) ₂ ] ₂ O(CH ₂ ) ₂ ; (CH ₂ ) ₃ O(CH ₂ ) ₄ O(CH ₂ ) ₃ ; (CH ₂ ) ₃ [O(CH ₂ ) ₂ ] ₂ O(CH ₂ ) ₃ ; (CH2) ₂ [O(CH ₂ ) ₂ ] ₃ O(CH ₂ ) ₂ ; (CH ₂ ) ₁₀ ; and (CH ₂ ) ₁₂ .

136. The herein provided compositions can bind single stranded RNA. The binding site can be an AU rich region. Thus, the binding site can comprise at least 5 contiguous bases of adenosine. As an example, the binding site can comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous bases of adenosine. Thus, the binding site can comprise at least 5 contiguous bases of uridine. As an example, the binding site can comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous bases of uridine. Thus, the binding site can comprise at least 5 contiguous bases of adenosine or uridine. As an example, the binding site can comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous bases of adenosine or uridine.

137. The herein provided compositions can bind a 16 base polyA-polyU RNA with a

Kd of less than or equal to 1 x 10" ⁶ , 1 x lO"?, 1 x 10" ⁸ , or 1 x 10"9.

138. The aminoglycoside of the provided composition can be any aminoglycoside known in the art or provided herein. Thus, in some aspects of the herein provided compositions, the aminoglycoside comprises neomycin. In some aspects, the aminoglycoside comprises a tobramycin.

139. In some aspects, the nucleic acid can comprise a nucleic acid capable of forming a duplex nucleic acid with RNA within 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 bases of the aminoglycoside binding site.

140. The amino groups on rings I, II and IV (neomycin, Figure 1) are necessary in stabilizing and recognizing various nucleic acid forms. Thus, in some aspects, the nucleic acid is covalently attached to the aminoglycoside at the 5-OH on the ribose of ring m.

141. The herein provided aminoglycoside conjugates can be conjugated to a nucleic acid at the 5' end of the nucleic acid or 3' end of the nucleic acid. Alternatively, the herein provided aminoglycoside conjugates can be covalently attached to a nucleotide and incorporated at any site within the nucleic acid.

142. The herein provided compositions can further comprise a minor groove binder. The minor groove binder can comprise any minor groove binder known in the art or provided herein. Thus, the minor groove binder can comprise Hoechst 33258 or polyamide.

, 143. Also provided herein is a method of increasing the affinity of a nucleic acid for a single-stranded RNA molecule comprising incubating an aminoglycoside conjugate provided herein with a single-stranded RNA molecule.

144. Also provided herein is a method of inhibiting a protein from interacting with a single-stranded RNA molecule comprising incubating an aminoglycoside conjugate provided herein with the single-stranded DNA.

4. Aminoglycosides

145. Aminoglycoside antibiotics are bactericidal drags that have been at the forefront of antimicrobial therapy for almost five decades. The past decade (1990—2000) saw a resurgence in aminoglycoside-based drag development as their chemistry/mechanism of action became better understood. This work, however, had almost exclusively focused on targeting RNA.

146. Aminoglycoside antibiotics (Figure 1 and Figure 2) are bactericidal agents that are comprised of two or more amino sugars joined in glycosidic linkage to a hexose nucleus (Chow CS, et al (1997) Chem Rev 97:1489). Though they exhibit a narrow toxic/therapeutic ratio, their broad antimicrobial spectrum, rapid bactericidal action, and ability to act synergistically with other drags makes them highly effective in the treatment of nosocomial (hospital acquired) infections (Kotra LP, et al (2000) J Urol 163:1076). They are clinically useful in the treatment of urinary tract infections (Santucci R, et al (2000) J Urol 163:1076), lower respiratory infections, bacteremias, and other superinfections by resistant organisms (Forge A, et al (2000) Audio Neurootol 5:3). Their greatest potential has been in combination drug regimens for the treatment

of infections that are difficult to cure with single agents and for use in patients who are allergic to other classes of drugs (Gerding D (2000) Infect Control Hosp Epidemiol 21 : S12). Aminoglycosides (Figures 1 and 2) contain a unique polyamine/carbohydrate structure, and have attracted considerable attention because of their specific interactions with RNA (Kaul M, et al (2003) J MoI Biol 326:1373). The bactericidal action of aminoglycosides is attributed to the irreversible inhibition of protein synthesis following their binding to the 30S subunit of the bacterial ribosome and thus interfering with the mRNA translation process. The miscoding causes membrane damage, which eventually disrupts the cell integrity, leading to bacterial cell death (Moazed D, et al (1987) Nature 327:389; Purohit P, et al (1994) Nature 370:659; Recht MI, et al D (1996) J MoI Biol 262:421; Miyaguchi H, et al (1996) Nucleic Acids Res 24:3700).

147. Aminoglycosides are a group of antibiotics that are effective against certain types of bacteria. Those which are derived from Streptomyces species are named with the suffix - mycin, while those which are derived from micromonospora are named with the suffix -micin. The aminoglycosides are polar-cations which consist of two or more amino sugars joined in a glycosidic linkage to a hexose nucleus, which is usually in a central position.

148. Aminoglycosides include: amikacin, apramycin, arbekacin, bambermycins, butirosin, dibekacin, dibekacin, dihydrostreptomycin, fortimicin, geneticin, gentamicins (e.g., gentamicin Cl, gentamicin CIa, gentamicin C2, and analogs and derivatives thereof), isepamicin, kanamycins (e.g. kanamycin A, kanamycin B, kanamycin C, and analogs and derivatives thereof), lividomycin, micronomicin, neamine, neomycins (e.g. neomycin B and analogs and derivatives thereof), netilmicin, paromomycin, ribostamycin, sisomicin, spectinomycin, streptomycin, streptonicozid, tobramycin, trospectomycin, and viomycin.

149. Examples of such aminoglycoside antibiotics include kanamycin (Merck Index 9th ed. #5132), gentamicin (Merck Index 9th ed. #4224), amikacin (Merck Index 9th ed. #A1) , dibekacin (Merck Index 9th ed. #2969), tobramycin (Merck Index 9th ed. #9193), streptomycin (Merck Index 9th ed. #8611/8612), paromomycin (Merck Index 9th ed. #6844), sisomicin (Merck Index 9th ed. #8292), isepamicin and netilmicin, all known in the art. The useful antibiotics include the several structural variants of the above compounds (e.g. kanamycin A, B and C; gentamicin A, Cl, CIa, C2 and D; neomycin B and C and the like). The free bases, as well as pharmaceutically acceptable acid addition salts of these aminoglycoside antibiotics, can be employed.

5. Major groove binders

150. A major groove binder is a composition or compound which can bind the major groove of duplex nucleic acid. It is understood that there are B-major groove binders which bind B-form duplex and A-major groove binders which binder A-form duplex.

151. It is understood that the disclosed compositions can have any major groove binder conjugated to it, as disclosed herein. The major groove binders disclosed below are exemplary only.

152. If a target gene contains a mutation that is the cause of a genetic disorder, then the herein provided method of preparing oligonucleotides is useful for mutagenic repair that may restore the DNA sequence of the target gene to normal. If the target gene is an oncogene causing unregulated proliferation, such as in a cancer cell, then the oligonucleotide is useful for causing a mutation that inactivates the gene and terminates or reduces the uncontrolled proliferation of the cell. The oligonucleotide is also a useful anti-cancer agent for activating a repressor gene that has lost its ability to repress proliferation. Furthermore, the oligonucleotide is useful as an antiviral agent when the oligonucleotide is specific for a portion of a viral genome necessary for proper proliferation or function of the virus. a) The Nucleic Acid Triplex

153. The biochemical access to a living organism's genetic information (stored in DNA) is based on specific protein-DNA interactions. Predictive chemical principles for protein- DNA recognition are still considered complex, despite the recent progress using biological selection methods (Greisman HA, et al (1997) Science 275:657-661; Wolfe SA, et al (2000) Annu Rev Biophys Biomol Struct 29:183; Choo Y, et al 1997) Cur Opin Struct Biol 7:117-125; Nagai K (1996) Curr Opin Struct Biol 6:53; Ellenberger T (1994) Curr Opin Struct Biol 4:12). Recognition of duplex DNA by small molecules (minor groove binders-polyamides) (Dervan PB (1986) Science 232:464; Dervan PB, et al (1999) Cur Opin Chem Biol 3:688; Gottesfeld JM, et al (2000) Gene Expr 9:77; Geierstanger BH, et al (1995) Annu Rev Biophys Biomol Stnuct 24:463; Wemmer DE (2000) Annu Rev Biophys Biomol Struct 29:439; Kopka ML, et al (1985) Proc Natl Acad Sci USA 82:1376-1380) and oligonucleotides (major groove binders-DNA triple helices) (Felsenfeld U, et al (1957) Biochim Biophys Acta 26:457; Moser HE, et al (1987) Science 238:645; Praseuth D, et al (1999) Biochim Biophys Acta 1489:181) are promising alternate approaches to a chemical solution for DNA recognition. Triple strand formation has also been exploited to facilitate the delivery and enhance the sequence specificity of DNA- cutting reagents (Moser HE, et al (1987) Science 238:645) (Francois J-C, et al (1989) Proc Natl

Acad ScfUSα"8 ^" 6:9702;'"Sfrøbel SA, et al (1988) J Am Chem Soc 110:7927) and drags (Praseuth D, et al (1999) Biochim Biophys Acta 1489:181; Sun J-S ₅ et al (1989) Proc Natl Acad Sci USA 86:9198). In addition, triple strand formation has been used to modify enzyme cutting patterns by selectively blocking enzyme binding sites in the major groove (Maher IE U, et al (1989) Science 245:725; Hanvey JC, et al (1989) Nucleic Acids Res 18:157). In short, appropriately designed and constructed third strand oligonucleotides that hybridize to targeted duplex domains can be used to control gene-expression, serve as artificial endonucleases in gene mapping strategies, dictate or modulate the sequence specificity of DNA-binding drags, and selectively alter the sites of enzyme activity. Ligands that increase the rates of association of a triplex forming oligonucleotide (TFO) to a target duplex thus have enormous potential in drug development and as tools for molecular biology.

154. Triple helix formation (see Figure 3 for H-bonding in different types of triple helical structures) has been the focus of considerable interest because of possible applications in developing new molecular biology tools as well as therapeutic agents (Frank-Kamenetskii MD, et al (1995) Annu Rev Biochem 64:65; Kool ET (1998) Ace Chem Res 31:502), and the possible relevance of H-DNA structures in biological systems (Moser HE, et al (1987) Science 238:645) (Wells RD, et al (1988) FASEB J 2:2939; Htun H, et al (1989) Science 243:1571; Htun H, et al (1988) Science 241 :1791). Intermolecular triplexes have aroused considerable interest as potential inhibitors of the expression of particular genes, since a sequence of either third-strand pyrimidines or purines, when 16-18 base pairs long, can be sufficient to be unique for recognition and binding to defined single sites in a genome (Praseuth D, et al (1999) Biochim Biophys Acta 1489:181).

155. A number of experiments have now been reported that demonstrate the feasibility of the concept (Grigoriev M, et al (1992) J Biol Chem 267:3389; Grigoriev M, et al (1993) Proc Natl Acad Sci USA 90:3501).

156. dA.dT + dT → dA-2dT (1)

157. Association of a third strand with a duplex, however, is a thermodynamically weaker and a kinetically slower interaction than duplex formation itself (Eq. 1) (Craig ME, et al (1971) J MoI Biol 62:383; Rougee M, et al (1992) Biochemistry 31:9269).

158. A number of intercalators, groove binders and polyamines have been used to stabilize triple helices (Escude C, et al (1996) Biochemistry 35:5735; Escude C, et al C (1995) J Amer Chem Soc 117:10212; Escude C, et al (1996) Chem Biol 3:57; Escude C, et al (1998) Proc Natl Acad Sci USA 95:3591; Kim SK, et al (1997) Biopolymers 42:101; Nguyen CH, et al

(l99&γrAm ^' ϋαem Sδe"ϊ20^50l; Tarui M, et al (1994) Biochem J 304 (1): 271; Wilson WD, et al (1994) J MoI Recognit 7:89; Cassidy SA, et al (1994) Biochemistry 33:15338; Wilson WD, et al (1993) Biochemistry 32:10614; Mergny JL, et al (1992) Science 256:1681; Choi S-D, et al (1997) Biochemistry 36:214; Scaria PV, et al (1991) J Biol Chem 266:5417; Mergny J-U, et al (1991) Nucleic Acids Res 19:1521; Latimer LIP ₅ et al (1995) Biochem Cell Biol 73:11; Lee JS, et al (1993) Biochemistry 32:5591; Denny WA (1989) Anticancer Drug Des 4:241; Pilch DS, (1993) J MoI Biol 232:926; Pilch DS, et al (1993) J Am Chem Soc 115:2565; Ren J, et al (2000) J Am Chem Soc 122:424; Strekowski U, et al (1996) J Med Chem 39:3980; Fox KR, et al (1996) Biochem Biophys Res Cominun 224:717; Fox KR, et al (1995) Proc Natl Acad Sci USA 92:7887; Haq I, et al (1996) J Amer Chem Soc 118:10693; Kan Y, et al (1997) Biochemistry 36:1461; Durand M, et al (1996) Biochemistry 35:9133; Durand M, et al (1994) J Biomol Struct Dyn 11:1191; Pilch DS, et al (1995) Biochemistry 34:16107; Pilch DS. et al (1994) Proc Natl Acad Sci USA 91 :9332; Durand M, et al (1992) J Biol Chem 267:24394; Antony T, et al (1999) Antisense Nucleic Acid Drug Dev 9:221; Basu HS, et al (1987) Biochem J 244:243; Musso M, et al (1997) Biochemistry 36:1441; Thomas TJ, et al (1996) Biochem J 319:591; Thomas I, et al (1993) Biochemistry 32:14068; Pallan PS, et al (1996) Biochem Biophys Res Commun 222:416; Nagamani D, et al (2001) Org Lett 3:103; Rajeev KG, et al (1997) J Org Chem 62:5169; Potaman VN, et al (1995) Biochemistry 34:14885; Mamyama A, et al (1997) Bioconjug Chem 8:3). The design of ligands that bind strongly to triple-helical structures and have a high discrimination between triplexes and duplexes opens new possibilities to control gene expression at the transcriptional level. There is a significant amount of high-resolution information on complexes of compounds that bind to both DNA and RNA by intercalation, and on compounds that bind in the DNA minor groove (Wemmer DE (2000) Annu Rev Biophys Biomol Struct 29:439; DervanPB (2001) Bioorg Med Chem 9:2215). Good models exist for proteins and peptides that bind in the major groove of DNA and RNA (Wolfe SA ₅ et al (2000) Annu Rev Biophys Biomol Struct 29:183; Nagai K (1996) Curr Opin Struct Biol 6:53).

(1) Antigene

159. In the antigene method, a triplex forming oligonucleotide (TFO) is wound round a double-stranded DNA chain of a target gene to form a triple-stranded chain. Because the amount of such target in cells is small, unlike the case of the anti-messenger method whose target is messenger RNA ₅ the antigene method which targets gene DNA is a more suitable method for clinical application, when stability and the like of oligonucleotides in the living body are taken

"into cbfaSfdbtationlToMMotfof the triple-stranded chain which is essential for the antigene method depends on the Hoogsteen bonding.

160. It has been shown that the polynucleotide polydT will bind to the polydA-polydT duplex to form a colinear triplex (Arnott, S & Seising E. (1974) J. Molec. Biol. 88, 509). The structure of that triplex has been deduced from X-ray fiber diffraction analysis and has been determined to be a colinear triplex (Arnott, S & Seising E. (1974) J. Molec. Biol. 88, 509). The polydT strand is bound in the parallel orientation to the polydA strand of the underlying duplex. The polydT-polydA-polydT triplex is stabilized by T-A Hoogstein base pairing between A in the duplex and the third strand of polydT. That interaction necessarily places the third strand, called a ligand, within the major groove of the underlying duplex. The binding site in the major groove is also referred to as the target sequence.

161. Similarly, it has been shown that polydG will bind by triplex formation to the duplex polydG-polydC, presumably by G-G pairing in the major helix groove of the underlying duplex, (Riley M., Mailing B. & Chamberlin M. (1966) J. Molec. Biol. 20, 359). This pattern of association is likely to be similar to the pattern of G-G-C triplet formation seen in fRNA crystals (Cantor C. & Schimmel P., (1980) Biophysical Chemistry vol I, p. 192-195).

162. Triplexes of the form polydA-polydA-polydT and polydC-polydG-polydC have also been detected (Broitman S., Im D. D. & Fresco J. R. (1987) Proc. Nat. Acad. Sci USA 84, 5120 and Lee J. S., Johnson D. A. & Morgan A. R. (1979) Nucl. Acids Res. 6, 3073). Further the mixed triplex polydCT-polydGA-polydCT has also been observed. (Parseuth D. et al. (1988) Proc. Nat. Acad Sci. USA 85, 1849 and Moser H. E. & Dervan P. B. (1987) Science 238, 645). These complexes, however, have proven to be weak or to occur only at acid PH.

163. Parallel deoxyribo oligonucleotide isomers which bind in the parallel orientation have been synthesized (Moser H. E. & Dervan P. E. (1987) Science 238, 645-650 and Rajagopol P. & Feigon J. (1989) Nature 339, 637-640). In examples where the binding site was symmetric and could have formed either the parallel or antiparallel triplex (oligodT binding to an oligodA- oligodT duplex target), the resulting triplex formed in the parallel orientation (Moser H. E. & Dervan P. E. (1987) Science 238, 645-650 and Praseuth D. et al (1988) PNAS 85, 1349-1353), as had been deduced from x-ray diffraction analysis of the polydT-polydA-polydT triplex.

164. Studies employing oligonucleotides comprising the unnatural alpha anomer of the nucleotide subunit, have shown that an antiparallel triplex can form (Praseuth D. et al. (1988) PNAS 85, 1349-1353). However, since the alpha deoxyribonucleotide units of DNA are inherently reversed with respect to the natural beta subunits, an antiparallel triplex formed by

aipna"θiigontici'edtϊaes"ϊlefces'§anly follows from the observation of parallel triplex formation by the natural beta oligonucleotides. For example, alpha deoxyribo oligonucleotides form parallel rather than antiparallel Watson-Crick helices with a complementary strand of the beta DNA isomer.

165. It has been demonstrated that a DNA oligonucleotide could bind by triplex formation to a duplex DNA target in a gene control region; thereby repressing transcription initiation (Cooney M. et. al. (1988) Science 241, 456). This was an important observation since the duplex DNA target was not a simple repeating sequence.

166. Triplex forming oligonucleotides (TFOs) are designed by scanning genomic duplex DNA and identifying nucleotide target sequences of greater than about 20 nucleotides having either about at least 65% purine bases or about at least 65% pyrimidine bases; and synthesizing said synthetic oligonucleotide complementary to said identified target sequence, said synthetic oligonucleotide having a G when the complementary location in the DNA duplex has a GC base pair, having a T when the complementary location of the DNA duplex has an AT base pair.

167. After identifying a DNA target with an interesting biological function, an oligonucleotide length must be chosen. There is typically a one to one correspondence between oligonucleotide length and target length. For example, a 27 base long oligonucleotide can be used to bind to a 27 base pair long duplex DNA target. Under optimal conditions, the stability of the oligonucleotide-duplex DNA interaction generally increases continuously with oligonucleotide length. Generally, a DNA oligonucleotide in the range of about 20 to 40 bases is used. For oligonucleotides in this range, the dissociation constants are in the range of about 10 ^"9 to 10 ^"8 molar.

168. Provided herein is a new method for designing synthetic oligonucleotides which will bind tightly and specifically to any duplex DNA target. When the target serves as a regulatory protein the method can be used to design synthetic oligonucleotides which can be used as a class of drug molecules to selectively manipulate the expression of individual genes.

(2) Triplex enhancement

169. In line with what is disclosed herein regarding nucleic acid modifications, one skilled in the art will recognize that a variety of synthetic procedures are available. Ih the preferred embodiment the oligonucleotides are synthesized by the phosphoramidite method, thereby yielding standard deoxyribonucleic acid oligomers.

TiCf. Mecul'ar modeling suggests that substitution of the non-hydrolyzable phosphodiester backbone in the oligonucleotide or elected sites may enhance the stability of the resulting triplex in certain instances. The phosphodiester analogues are more resistant to attack by cellular nucleases. Examples of non-hydrolyzable phosphodiester backbones are phosphorothioate, phosphoroselenoate, methyl phosphate, phosphotriester and the alpha enantiomer of naturally occurring phosphodiester. These non-hydrolyzable derivatives of the proposed oligonucleotide sequences can be produced, with little alteration of DNA target specificity.

171. Backbone modification provides a practical tool to "fine tune" the stability of oligonucleotide ligands inside a living cell. For example, oligonucleotides containing the natural phosphodiester linkage are degraded over the course of 1-2 hours in eukaryotic cells, while the non-hydrolyzable derivatives appear to be stable indefinitely.

172. Oligonucleotide hybrids provide another method to alter the characteristics of the synthetic oliogonucleotides. Linkers can be attached to the 5' and/or 3' termini of the synthetic oligonucleotide. The linkers which are attached to the 5' terminus are usually selected from the group consisting of a base analogue with a primary amine affixed to the base plane through an alkyl linkage, a base analogue with a sulfhydryl affixed to the base plane through an alkyl linkage, a long chain amine coupled directly to the 5' hydroxyl group of the oligonucleotide and a long chain thiol coupled directly to the 5' hydroxyl group of the oligonucleotide. The linker on the 3' terminus is usually a base analogue with a primary amine affixed to the base plane through an alkyl linkage or a base analogue with a sulfhydryl affixed to the base plane through a alkyl linkage. Affixation of a primary amine linkage to the terminus does not alter oligonucleotide binding to the duplex DNA target.

(a) Hybrids

173. As discussed through out this application, once a linkage has been attached to the synthetic oligonucleotide a variety of modifying groups can be attached to the synthetic oligonucleotide. The molecules which can attach include intercalators, groove-binding molecules, cationic amines or cationic polypeptides. The modifying group can be selected for its ability to damage DNA. For example, the modifying group could include catalytic oxidants such as the iron-EDTA chelate, nitrogen mustards, alkylators, photochemical crosslinkers such as psoralin, photochemical sensitizers of singlet oxygen such as eosin, methylene blue, acridine orange and 9 amino acridine and reagents of direct photochemical damage such as ethidium and various pyrene derivatives.

174:" 'It ii'pόMWe tδ improve affinity of an oligonucleotide by binding a DNA intercalator or the like to its 5'- or 3'-end or to increase its incorporation into cells by binding thereto a fat-soluble compound such as cholesterol. Acridine or a derivative thereof can be cited as an example of the DNA intercalator (WO 92/20698).

175. Thus, an "aminolink" coupling can be used to affix acridine isothiocanate to the provided oligonucleotides. The duplex binding affinity of the oligonucleotide-dye hybrid is approximately 100-fold greater than the oligonucleotide binding affinity. Also provided is the binding of eosin isothiocyanate to oligonucleotides. Since eosin isothiocyanate cleaves the DNA helix upon irradiation this hybrid oligonucleotide cuts the helix at its binding site when irradiated. This hybrid-oligonucleotide is useful for identifying the oligonucleotide binding site both in vitro and in vivo and potentially can be used as a therapeutic tool for selective gene target destruction.

176. Photochemical reactivity is also achieved by affixation of psoralin derivatives to oligonucleotides through a 5' linkage. Psoralin binds covalently to DNA after irradiation, and as a consequence is a potent cytotoxic agent. Thus, photochemical reactivity, with oligonucleotide sensitivity provides a tool to direct the toxic psoralin lesion to the oligonucleotide target site.

177. Similar oligonucleotide coupling is used to target toxic chemical reactivity to specific DNA sequences. Examples include catalytic oxidants such as transition metal chelates and nucleases. Photochemical reactivity and/or toxic chemical agents can be used to permanently inhibit gene expression.

178. In addition to chemical reactivity, modifications of oligonucleotides alter the rate of cellular uptake of the hybrid oligonucleotide molecules. Terminal modification provides a useful procedure to modify cell type specificity, pharmacokinetics, nuclear permeability, and absolute cell uptake rate for oligonucleotide ligands.

(b) Analogues

179. As discussed herein for nucleic acids, modified base analogues provide another means of altering the characteristics of the synthetic oligonucleotide. Although a purine rather than a pyrimidine, X is identical to T with respect to its capacity to form hydrogen bonds. Molecular modeling has shown that substitution of X for T in the above oligonucleotide design procedures, results in a modified triplex that is much more stable. The increased stability is due principally to enhanced stacking and to an enhancement of phosphodiester backbone symmetry within the ligand. Examples of base substitutions for T are X, I and halogenated X and I. G can be replaced by halogenated G. Furthermore, the 2' furanose position on the base can have a non-

ύhtogM'bWKy grtiwp MlJfStItUIiOn. Examples of non-charged bulky groups include branched alkyls, sugars and branched sugars. In the preferred embodiment at least one base is substituted.

(3) Duplex triplex DNA/RNA

180. Triple-helical DNA consists of a Watson-Crick duplex with a third strand bound within the major groove, forming Hoogsteen hydrogen bonds. Nucleic acid bases can form specific hydrogen bonds with purine (A, G) bases already engaged in Watson-Crick hydrogen bonding interactions with complementary bases.

181. These non- Watson-Crick hydrogen bonds were first observed by Hoogsteen in co- crystals of adenine and thymine derivatives and are often referred to as Hoogsteen and reverse Hoogsteen hydrogen bonds. Triplex-forming oligonucleotides (TFOs) that bind to DNA in a sequence-specific manner can provide an effective way to selectively modulate gene expression via transcriptional repression, mutagenesis and recombination. Binding of a TFO requires the presence of a relatively long and uninterrupted homopurinerhomopyrimidine tract in DNA to ensure optimal stability and specificity of the triple helical complex. Studies in cell-free systems have shown that under optimal conditions (i.e., presence of Mg2+, appropriate pH and limited concentrations of monovalent cations), TFOs bind to homopurine (G, A) target sequences with dissociation constants (Kd) that are comparable to those of many DNA binding proteins such as transcription regulatory factors, and inhibit transcription in cell-free systems: This strategy has proven to be successful in various experimental models, including living cells and animals, and can provide the means for design of novel gene-targeted therapeutics.

182. However, triple-helical complexes formed by oligonucleotides with double- helical DNA are less stable than the double-helical complexes formed by the same oligonucleotides bound to complementary single-stranded sequences.

(4) Triplex forming oligonucleotides

183. Triplex forming functional nucleic acid molecules are molecules that can interact with either double-stranded or single-stranded nucleic acid. When triplex molecules interact with a target region, a structure called a triplex is formed, in which there are three strands of DNA forming a complex dependant on both Watson-Crick and Hoogsteen base-pairing. Triplex molecules are preferred because they can bind target regions with high affinity and specificity. It is preferred that the triplex forming molecules bind the target molecule with a k _d less than 10 ^"6 , 10 ^"8 , lO ^"10 , or 10 ^"12 . Representative examples of how to make and use triplex forming molecules to bind a variety of different target molecules can be found in the following non-limiting list of

ϋmteS SMtfelφatfiϊtsf 5;'176^96, 5,645,985, 5,650,316, 5,683,874, 5,693,773, 5,834,185, 5,869,246, 5,874,566, and 5,962,426.

184. Triplex forming oligonucleotides (TFO) have been used for targeted mutagenesis (see U.S. Patent 5,874,566, herein incorporated by reference in its entirety for its teaching of TFO for targeted mutagenesis). The oligonucleotides herein described can be used alone or in combination with other mutagenic agents. As used herein, two agents are said to be used in combination when the two agents are co-administered, or when the two agents are administered in a fashion so that both agents are present within the cell or serum simultaneously. An agent suitable for co-administration is psoralen-linked oligonucleotides as described in PCT/US94/07234 by Yale University.

185. The oligonucleotides herein described can further be used to stimulate homologous recombination of a exogenously supplied, DNA fragment, into a target region. Specifically, by activating cellular mechanisms involved in DNA synthesis, repair and recombination, the oligonucleotides herein described can be used to increase the efficiency of targeted recombination.

186. In targeted recombination, a triplex forming oligonucleotide is administered to a cell in combination with a separate DNA fragment which minimally contains a sequence complementary to the target region or a region adjacent to the target region, referred to herein as the recombination fragment. The recombination fragment can further contain nucleic acid sequences which are to be inserted within the target region. The co-administration of a triplex forming oligonucleotide with the recombination fragment increases the frequency of insertion of the recombination fragment within the target region when compared to procedures which do not employ a triplex forming oligonucleotide.

(5) Foldback FTOs

187. Also provided are foldback triplex-forming oligonucleotides (FTFOs) comprising sequences in which one or more nucleotides are replaced with an abasic moiety, i.e., a moiety without a base.

188. The FTFOs provided herein can comprise a duplex-forming region, a triplex- forming region, and a linker region. The duplex-forming region hybridizes to the target nucleic acid through normal Watson-Crick bonding and the triplex-forming region folds back upon the duplex thereby formed to form a triplex by Hoogsteen bonding to the duplex. Typically, hybridization of the third strand (be it either a complex of three separate nucleic acid strands or the foldback triplex-forming approach) is to a homopurine region of one of the stands involved

in the Watson-Cnck dupexrAccordingly, art recognized FTFOs generally target homopurine sequences only. The FTFOs of the present invention, however, can also target polypurine sequences having several interspersed pyrimidine nucleotides. They can accomplish this by including in the triplex-forming region one or more abasic linkers positioned such that, during triplex formation, the abasic linkers match up against the interspersed pyrimidine nucleotide of the target nucleic acid, thereby resulting in the triplex-forming region "skipping over" the pyrimidine nucleotide as Hoogsteen-type hydrogen bonds are formed. FTFOs with abasic linkers have been described in U.S. Patent 5,693,773, herein incorporated by reference in its entirety for its teaching of FTFOs. For example, the linkers can be used to bridge a triplex binding region and an aminoglycoside dimer binding region.

189. The duplex forming region of the FTFOs provided herein is sufficiently complementary to the target nucleic acid (in the Watson-Crick sense) to hybridize to the target under the conditions (e.g., pH and temperature) of interest. The triplex-forming region is generally complementary (in the Hoogsteen sense) to the duplex forming region, being the mirror image of the duplex forming region. The linker region connects the duplex-forming region and the triplex-forming region, allowing the oligonucleotide to fold upon itself, and comprises, for example, a short nucleotide sequence (e.g., a pentanucleotide) or some other non- nucleotide substituent such as ethylene glycol.

190. The number of abasic linkers that can be incorporated into the oligonucleotides disclosed herein depends on the length of the triplex-forming region. The longer the triplex forming region, the more abasic linkers that can be incorporated. When the triplex-forming region is short (e.g., about 10 nucleotides in length), only one or two abasic linkers can be incorporated and still maintain the triplex-forming ability of the oligonucleotide. When the triplex-forming region is long (about 30 nucleotides or more), 4 or 5 abasic linkers can be used.

191. Foldback triplex-forming oligonucleotides provided herein are useful tools for gene modulation and have both in vitro and in vivo utilities. Because FTFOs of the present invention can inhibit gene expression, in vitro they can be used as a convenient alternative to the laborious technique of deletion mutation for the determination of gene function. The importance of this use is easily appreciated when one realizes that the biological function of most known genes was determined by deletion mutation.

192. In vivo, the FTFOs of the present invention are therapeutically useful for treating a wide variety of maladies ranging from pathogen caused diseases to aberrant expression of endogenous nucleic acids. By targeting the pathogenic or aberrant nucleic acid, administration of

the ^' provided 'FTFOs cWϊfihMit expression of the nucleic acid, thereby preventing further adverse effects.

(6) Circular TFO

193. Also provided are single-stranded circular TFOs, which by nature of the circularity of the oligonucleotide and the domains present on the oligonucleotide, are nuclease resistant and bind with strong affinity and high selectivity to their targeted nucleic acids. Circular TFOs have been described in U.S. Patent 5,683,874, herein incorporated by reference in its entirety for its teaching of Circular TFOs.

194. As an example, the provided single-stranded circular TFOs can have at least one Hoogsteen parallel binding (P) domain and at least one Watson-Crick anti-parallel binding (AP) domain and have a loop domain between each binding domain, so that a circular oligonucleotide is formed. As another example, the single stranded circular oligonucleotides can have at least one of a P domain, a Hoogsteen anti-parallel (HAP) domain and an AP domain and a loop domain between each binding domain. Ih circular TFOs having one binding domain, the loop domain is between the ends of the binding domain so that a circular oligonucleotide is formed. Moreover, each P, HAP and AP domain exhibits sufficient complementarity to bind to one strand of a defined nucleic acid target with the P domain binding to the target in a parallel manner and the HAP and AP domains binding to the target in an anti-parallel manner. b) Noncovalent triplex interactions with other molecules

(1) Effect of Neomycin on a Polynucleotide Triplex

195. Neomycin selectively stabilizes DNA triplex without affecting the duplex (Arya DP, et al (2001) J Am Chem Soc 123:11093; Arya DP, et al (2001) J Am Chem Soc 123:5385; Arya DP, et al (2000) Bioorganic Med Chem Lett 10:1897), (Arya DP, et al (2003) J Am Chem Soc 125:3733). Increasing the molar ratios of neomycin from 0-25 μM, Vά _b (ratio drug [neomycin]/base triplet)=1.67, increases the triplex melting point by nearly 25°C, whereas the duplex is virtually unaffected (Figure 4).

(2) Thermal Denaturation Studies with Poly(dA).2poly(dT) in the Presence of Other Aminoglycosides and Diamines

196. Thermal analysis of poly(dA)»2poly(dT) in the presence of other aminoglycosides is shown as a bar graph in Figure 5. At high concentrations (r _db =0.66-1.67, Figure 5), most aminoglycosides with five or more amines are able to stabilize the triple helix (increasing δT _{m3→ 2} , without significantly affecting the δT _m2→ i values). The difference between the effectiveness of paromomycin and neomycin is quite remarkable. The structural difference

βe'Weeffme'twb isVposifively charged amino group (present in neomycin), replacing a neutral hydroxyl (present in paromomycin). This leads to a difference of 10°C in T _m3 -» ₂ values (r^ =0.66) and a difference of 16°C at r _db =1.67 (Arya DP, et al (2001) J Am Chem Soc 123:5385). At lower concentration of antibiotics (r _<3b =0.26), paromomycin has little effect on the stability of the triplex. Lividomycin, a paromomycin analog with a polyhydroxy hexose tether, is slightly less effective than paromomycin in increasing T _m3 → ₂ values under these conditions.

(3) Stabilization of DNA Triple Helix Poly(dA)_2poly(dT) by

Other DNA Groove Binders

197. In order to assess how neomycin compares to other ligands in stabilizing triplexes, thermal denaturation analyses of poly(dA)»2poly(dT) triplex in the presence of previously studied DNA minor groove binders (Figure 6) has also been performed (Figure 7). A comparison with groove binders, shown in Figure 8, indicates that neomycin is much more active than the minor groove binders (berenil, DOC, DODC, DAPI, Hoechst 33258, Hoechst 33342). The minor groove binders previously studied have little preference for triple helix (berenil, distamycin and Hoechst dyes). Most groove binders stabilize the duplex as well as the triplex (Hoechst, berenil, distamycin) and some even destabilize the triplex (berenil, distamycin). The groove-binding ability of neomycin was extremely unique and presented a novel mode of triplex recognition. Neomycin, as opposed to other groove binders, differentiated the triplex grooves from those present in the duplex (Figure 7).

(4) Thermodynamics of Drug Binding to the DNA Triplex (ITC)

198. An ITC-derived thermodynamic profile for neomycin binding to 12-mer intramolecular DNA triplex gave a binding constant of 2.OxIO ⁵ MT ¹ (Figure 8 and Table 1). The complexation is enthalpy-driven (81%), with little entropic contributions. The binding is salt- dependent, with higher salt leading to a decrease in the association equilibrium constant. A much higher binding constant of neomycin is observed with other nucleic acids-RNA triplex/DNA tetraplex.

199. Table 1. ITC-derived thermodynamic profiles for the binding of neomycin to 5'- dA ₁₂ -x-dT _ir x-dT ₁₂ -3' triple helix in 10 mM sodium cacodylate 0.5 mM EDTA, 150 mM KCl, pH 6.8 at 20°C

(5) CD/Molecular Modeling

200. Neomycin has a marked preference for binding to the larger Watson-Hoogsteen (W-H) groove of the triplex (Arya DP, et al (2003) J Am Chem Soc 125:3733). Ring 1/R amino groups and Ring IV amines were proposed to be involved in the recognition process. CD/ITC studies indicate a five base triplet/drug binding site. The novel selectivity of neomycin was shown to be a function of its charge and shape complementarity to the triplex W-H groove (Figure 9) (Arya DP, et al (2003) J Am Chem Soc 125:3733).

201. Neomycin is the first molecule demonstrated to selectively stabilize DNA triplex structures that include polynucleotides, small homopolymer, as well as mixed base triplexes (Arya DP, et al (2003) J Am Chem Soc 125:3733). This stabilization is based on neomycin's ability to bind triplexes in the groove with high affinity (based on viscometric and ITC titrations). Modeling/physicochemical results indicate a further preference of neomycin binding to the larger W-H groove. These findings can contribute to the development of a new series of triplex-specific (DNA/RNA and hybrid) ligands, which can contribute to either antisense or antigene therapies.

(6) DNA.RNA Hybrids

202. RNA»DNA hybrid duplexes are the primary targets for important enzymes that include ribonuclease-H and reverse transcriptase (Kohlstaedt LI, et al (1992) Science 256:1783; Stein CA, et al (1988) Cancer Res 48:2659). Stable RNA.DNA triplexes normally adopt an A- type conformation and have been shown to inhibit RNA polymerase (Morgan AR, et al (1968) J MoI Biol 37:63), DNAase-I, and RNAse (Murray NL, et al (1973) Can J Biochem 51 :436). Only six of the eight possible combinations of triplexes are stable under physiological conditions (Han H, et al (1993) Proc Natl Acad Sci USA 90:3806; Wang 5, et al (1995) Nucleic Acids Res 23:1157). Stabilization of poly(rA).2poly(dT) and 2poly(rA).poly(dT) triplexes can only be achieved under molar salt conditions (Riley M, et al (1966) J MoI Biol 20:359). Since these two triplexes cannot be studied under the lower salt conditions used in competition dialysis assay, the effect of neomycin on these two triplexes was examined using UV and CD thermal denaturation studies. Neomycin stabilizes the hybrid poly(rA).poly(dT) duplex (Arya DP, et al (2001) J Am Chem Soc 123:11093), and even induce poly(rA)»2poly(dT) triplex formation (Arya DP, et al (2001) J Am Chem Soc 123:11093), much more effectively than previously reported ligands (Pilch DS. et al (1994) Proc Natl Acad Sci USA 91:9332). The effect of aminoglycosides on hybrid duplex and triplex structures showed that almost all aminoglycosides stabilized the hybrid poly(dA)«poly(dT) duplex (see Figure 10). It is noteworthy that formation of these triple helices

require molar salt in the absence of the drug, whereas micromolar neomycin concentration can induce the triplex formation. There is a high binding constant (10 M ^" ) for aminoglycoside binding to small RNA»DNA hybrids.

6. Minor groove binders

203. A minor groove binder is a composition or compound which can bind the minor groove of duplex DNA. It is understood that there are B-minor groove binders which bind the minor groove of b-form duplex and A-minor groove binders which bind the minor groove of A- form duplex.

204. It is understood that the disclosed compositions can have any minor groove binder conjugated to it, as disclosed herein. The minor groove binders disclosed below are exemplary only.

205. Minor groove recognition relies on van der Waals' contacts, hydrogen bonds, Coulombic attraction and intrinsic properties of the DNA such as flexibility, hydration and electrostatic potential. Successful minor groove binding ligands typically consist of heterocyclic units such as pyrrole or imidazole groups linked by amides. The flexibility of the single bonds between the heterocyclic groups and the amide linkages is crucial to successful minor groove recognition since the ligand is able to adopt a twist that matches the helical winding of the DNA, thereby permitting the ligand to maintain contact with the DNA over the foil length of its recognition site.

206. Two thoroughly studied minor groove binders (MGBs) are Hoechst 33258 (Hoechst) and DAPI, which bind preferentially at AT-rich regions of B-DNA. Also disclosed are minor groove binders, such as polyamides, that preferentially bind GC-rich regions. a) DNA-Selective Hoechst Dyes

207. The bisbenzimide dyes — Hoechst 33258, Hoechst 33342 and Hoechst 34580 are cell membrane-permeant, minor groove-binding DNA stains that fluoresce bright blue upon binding to DNA. Hoechst 33342 has slightly higher membrane permeability than Hoechst 33258, but both dyes are quite soluble in water (up to 2% solutions can be prepared) and relatively nontoxic. Hoechst 34580 has somewhat longer-wavelength spectra than the other Hoechst dyes when bound to nucleic acids. These Hoechst dyes, which can be excited with the UV spectral lines of the argon-ion laser and by most conventional fluorescence excitation sources, exhibit relatively large Stokes shifts (spectra) (excitation/emission maxima -350/460 nm), making them suitable for multicolor labeling experiments. The Hoechst 33258 and Hoechst 33342 dyes have complex, pH-dependent spectra when not bound to nucleic acids, with a much higher

fluorescence quantum ^' yield a ^' fpH 5 than at pH 8. Their fluorescence is also enhanced by surfactants such as sodium dodecyl sulfate (SDS). These dyes appear to show a wide spectrum of sequence-dependent DNA affinities and bind with sufficient strength to poly(d(A-T)) sequences that they can displace several known DNA intercalators. They also exhibit multiple binding modes and distinct fluorescence emission spectra that are dependent on dye:base pair ratios. Hoechst dyes are used in many cellular applications, including cell-cycle and apoptosis studies (Section 15.4, Section 15.5) and they are common nuclear counterstains (Section 8.6). Hoechst 33258, which is selectively toxic to malaria parasites, is also useful for flow-cytometric screening of blood samples for malaria parasites and for assessing their susceptibility to drugs; however, some of the SYTO dyes disclosed herein are likely to provide superior performance in these assays.

208. The Hoechst 33258 and Hoechst 33342 dyes are available as solids (H1398, H1399), as guaranteed high-purity solids (FluoroPure Grade; H21491, H21492) and, for ease of handling, as 10 mg/mL aqueous solutions (H3569, H3570). The Hoechst 34580 dye is available as a solid (H21486). b) AT-Selective DAPI

209. DAPI (4',6-diamidino-2-phenylindole) shows blue fluorescence (photo) upon binding DNA and can be excited with a mercury-arc lamp or with the UV lines of the argon-ion laser. Like the Hoechst dyes, the blue-fluorescent DAPI stain apparently associates with the minor groove of dsDNA (Figure 8.30), preferentially binding to AT clusters; there is evidence that DAPI also binds to DNA sequences that contain as few as two consecutive AT base pairs, perhaps employing a different binding mode. DAPI is thought to employ an intercalating binding mode with RNA that is AU selective.

210. The selectivity of DAPI for DNA over RNA is reported to be greater than that displayed by ethidium bromide and propidium iodide. Furthermore, the DAPI-RNA complex exhibits a longer-wavelength fluorescence emission maximum than the DAPI-dsDNA complex (-500 nm versus ~460 nm) but a quantum yield that is only about 20% as high.

211. Binding of DAPI to dsDNA produces an ~20-fold fluorescence enhancement, apparently due to the displacement of water molecules from both DAPI and the minor groove. Although the Hoechst dyes may be somewhat brighter in some applications, their photostability when bound to dsDNA is less than that of DAPI. In the presence of appropriate salt concentrations, DAPI usually does not exhibit fluorescence enhancement upon binding to ssDNA or GC base pairs. However, the fluorescence of DAPI does increase significantly upon

bϊridϊiϊ'g't'o'ddtfefgέϊltsf dextrafϊ sulfate, polyphosphates and other polyanions. A review by Kapuscinski discusses the mechanisms of DAPI binding to nucleic acids, its spectral properties and its uses in flow cytometry and for chromosome staining. DAPI is an excellent nuclear counterstain, showing a distinct banding pattern in chromosomes (Section 8.6, photo), and we have included it in our Cytological Nuclear Counterstain Kit (C7590, Section 8.6). DAPI is quite soluble in water but has limited solubility in phosphate-buffered saline. c) Others

212. It has been previously shown that distamycin A binds to the minor groove of B- form dsDNA (Zimmer C. and Wahnert,U. (1986) Prog. Biophys. MoI. Biol., 47, 31-112). Distamycin A has been shown to preferably bind to DNA duplex tracts containing a 5 bp A-T tract (Kopka MX., Yoon,C, Goodsell,D., Pjura,P. and Dickerson,R.E. (1985) Proc. Natl Acad. Sci. USA, 82, 1376-1380). Netropsin, on the other hand, preferentially binds to a DNA duplex tract containing a 4 bp A-T tract (Kopka M.L., Yoon,C, Goodsell,D., Pjura,P. and Dickerson,R.E. (1985) Proc. Natl Acad. Sci. USA, 82, 1376-138Q).

213. Among minor groove binders, the N-methylpyrrole carboxamide-containing antibiotics netropsin and distamycin bound to DNA with very pronounced AT specificity, as expected. More interestingly the dye Hoechst 33258, berenil and a thiazole-containing lexitropsin elicited negative reduced dichroism in the presence of GC-rich DNA which is totally inconsistent with a groove binding process. These three drugs share with DAPI the property of intercalating at GC-rich sites and binding to the minor groove of DNA at other sites. (Bailly, C, et al. 1992. Drug - DNA sequence-dependent interactions analysed by electric linear dichroism. Journal of Molecular Recognition. 5:4 (155-171).

214. 4-[(3-Methyl-6-(benzothiazol-2-yl)-2,3-dihydro-(benzo-l,3- thiazole)-2- methylidene)]-l-methyl-pyridinium iodide (BEBO) is an asymmetric monovalent cyanine dye that binds in the minor groove of double-stranded (ds)DNA. (Bengtsson, M, et al. A new minor groove binding asymmetric cyanine reporter dye for real-time PCR. Nucleic Acids Res. 2003 April 15; 31(8): e45).

215. Similarly to that of DAPI and Hoechst, the binding of BEBO to ρoly(dG-dC) ₂ is dominated by intercalation, and BEBO has a distinct preference for poly(dA-dT) ₂ compared to poly(dG-dC) ₂ .

216. As judged from the linear and circular dichroism studies, the benzoxazole derivative BOXTO exhibited straightforward minor groove binding both to poly(dA-dT) ₂ and calf thymus DNA (ctDNA), whereas the benzothiazole derivative BETO showed a more

heterogeneous binding to ^' the latter DNA, also with a greater tendency for aggregation. (Karlsson, HJ, et al. Groove-binding unsymmetrical cyanine dyes for staining of DNA: syntheses and characterization of the DNA-binding. Nucleic Acids Res. 2003 November 1; 31(21): 6227- 6234).

7. Exemplary schemes

217. For example, general forms of the disclosed compositions which can be used to bind B-form duplex or A-form duplex or single stranded nucleic acid are provided in the general schemes below. It is understood that the synthetic routes provided herein with what is known are capable of producing any of the disclosed combinations of, for example, aminoglycoside dimers, aminoglycoside conjugates with ODNs or other major groove binders or minor groove binders. For example, all aminoglycosides have alcohols and amines which can selectively be protected as described herein, for example, with a BOC protecting group and the the isothiocyanate can function as a general linking system. In each case, for example, a different alcohol may be chosen for linkage with the aminoglycoside but this can be determined for each aminoglycosdie as disclosed herein. Likewise, all of the molecules can be added to the 3' end of an ODN or nucleoside via the disclosed routes, typically the ODN would simply have to be removed from the column to free up the 3' OH. This can be done while maintaining the 5' protecting group. Similarly, any minor groove binder can be attached to either a linker or an ODN or an aminoglycoside as described herein, by for example, amine linkage through the isothiocyanate attached to the aminoglycoside.

218. It is understood that for schemes 1-7 below exemplary embodiments of aminoglycosides, linkers, minor groove binders, major groove binders, ODNS and other molecules which can be conjugated, but any example of any of these molecules disclosed herein or otherwise known can also be used in the schemes, even if they are not specifically recited in the schemes below.

a, b, c or

of

221. Exemplary Aminoglycosides: amikacin, apramycin, arbekacin, bambermycins, butirosin, dibekacin, dibekacin, dihydrostreptomycin, fortimicin, geneticin, gentamicins, isepamicin, kanamycins, lividomycin, micronomicin, neamine, neomycins, netilmicin, paromomycin, ribostamycin, sisomicin, spectinomycin, streptomycin, streptonicozid, tobramycin, trospectomycin, and viomycin.

222. MGB = Exemplary: distamycin, berenil, bis(benzimidazoles), DODC, DAPI, sequence specific polyamides (Kielkopf, C. L.; White, S.; Szewczyk, J. W.; Turner, J. M.; Baird, E. E.; Dervan, P. B.; Rees, D. C. Science 1998, 282, 111-115).

224. X = CH2 orNH

225. R ¹ = H, OH, or (CH2)nOH

226. R" = H, NH2, or (CH2)nNH2

Nucleic acid

227. X = PO ₂ or modified backbone

228. Y = nucleobase or modified nucleobase

229. Z = O ₅ S, CH ₂

230. R = H, OH, halogen, 0-,S-, N-alkyl/alkenyl/alkynyl

Scheme 5

ODN- linker -AG

MGB

ODN- linker ^■ AG Linker MGB

ODN- linker "MGB Linker -AG

MGB=minor groove binder-linked to a terminal CH2,-O-, S-, NH, NHCSNH, NHC0NH,C0NH,NHC0, or COS-

AG=aminoglycoside-linked to a terminal CH2-, 0-, S-, -NH, -NHCSNH, -NHC0NH,-C0NH,-NHC0, or -COS-

ODN=Oligonucleotide-linked to a terminal CH2.-0-, S-, NH, NHCSNH, NHCONH ₃ CONH ₅ NHCO ₃ COS, or OPO3-

231. Scheme 5 is a scheme for linking an oligonucleotide with an aminoglycoside and a minor grove binder

Scheme 6

MGB- linker-L -AG

AG=aminoglycoside-linked to a terminalO-, S-, NH, NHCSNH, NHCONH,CONH,NHCO, COS,

MGB=minor groove binder-linked to a terminal O-, S-, NH ₅ NHCSNH, NHCONH,CONH,NHCO, COS,

232. Scheme 6 is a scheme for linking an aminoglycoside and a minor grove binder

Scheme 7

ODN- linker-L -AG

AG=aminoglycoside-linked to a terminal CH2-, 0-, S-, -NH, -NHCSNH, -NHCONHrCONH ₅ -NHCO, -COS,

ODN=01igonucleotide-linked to a terminal CH2,-0-, S-, NH, NHCSNH, NHC0NH,C0NH,NHC0, COS,OPO3-

L

233. Scheme 7 is a scheme for linking an Oligonucleotide and an aminoglycoside

234. Shown above are two examples of polyamide (MGB)-neomycin(AG) conjugate. Polyamides preferably bind GC -rich regions in minor grooves. Other minor groove binders include distamycin, berenil, bis(benzimidazoles), DODC, DAPI, sequence specific polyamides (see Kielkopf, C. L.; White, S.; Szewczyk, J. W.; Turner, J. M.; Baird, E. E.; Dervan, P. B.; Rees, D. C. Science 1998, 282, 111-115).

8. Aminoglycosides and Nucleic Acids: The Attraction for RNA

235. Aminoglycosides have shown to bind to various RNA molecules. These include the 5 '-untranslated region of thymidylate synthase mRNA (Tok JBH, et al (1999) Biochemistry 38:199), both Rev response element and transactivating response element RNA motifs (Zapp ML, et al (1993) Cell 74:969; Hermann T, et al (1998) Biopolymers 48:155; Mei H-Y, et al (1995) Bioorg Med Chem Lett 5:2755) of HIV-I ₅ a variety of catalytic RNA molecules such as group I introns (Chow CS, et al (1997) Chem Rev 97:1489; von Ahsen U, et al (1993) Science 260:1500), ribonuclease P RNA (Mikkelsen NE, et al (1999) Proc Natl Acad Sci USA 96:6155), hairpin ribozyme (Walter F, et al (1998) Biochemistry 37:14195; Eamshaw DJ, et al (1998) Nucleic Acids Res 26:5551), hammerhead ribozyme (Kotra LP ₅ et al (2000) J Urol 163:1076) (Clouet-d'Orval B, et al (1995) Biochemistry 34:11186; Stage TK, et al (1995) RNA 1:95), and hepatitis delta virus ribozyme (Chia JS, et al (1997) J Biomed Sci 4:208; Rogers J, et al (1996) J MoI Biol 259:916). Aminoglycosides' binding to HIV-I RNA molecules have been shown to

prevent binding of the cognate viral proteins Tat and Rev to TAR (Mei H-Y, et al (1997) Bioorg Med Cham 5:1173) and RRE (Zapp ML, et al (1993) Cell 74:969), respectively. The glucose residues present in glycosylated DNA render the DNA inaccessible for enzymes, and thus help the pathogen escape degradation by host restriction enzymes (van Leeuwen F, et al (1998) Anal Biochem 258:223; Gao Y-g, et al (1997) J Amer Chem Soc 119:1496). A large number of different RNA structures that aminoglycosides bind have been identified. The reason for this RNA-centered development was understandable: aminoglycosides exhibit their antibacterial action through rRNA binding and show high affinity binding (K _d in the nanomolar range) to such RNAs.

9. DNA vs RNA Recognition

236. RNA recognition has proven to be more challenging than DNA recognition by small molecules.Recognition of DNA«RNA hybrids by small molecules was virtually unexplored at the beginning of this century (Ren J, et al (2001)). DNA-based intercalators and groove binders were the first to be examined for RNA recognition. These approaches met with limited success, due in large part to the different 3-D structures of functional RNA molecules. Sequence-specific RNA recognition has more similarities to recognition principles used in targeting proteins than to DNA duplexes. As with proteins, a distribution of charged pockets can provide a 3-D pattern that can be targeted specifically by compounds exhibiting structural electrostatic complementarity. Aminoglycosides have been shown to provide complementary scaffolds where the positively charged ammonium groups displace several Mg ²⁺ ions from their RNA binding sites (Tor Y, et al (1998) Chem Biol 5: R277; Hermann T (2000) Angew Chem Int Ed Engl 39:1890; Hermann T, et al (1998) Biopolymers 48:155; Hermann T, et al (1998) J MoI Biol 276:903; Hermann T, et al (1998) Curr Opin Biotechnol 9:66; Hermann T, et al (1999) J Med Chem 42:1250; Henry CM (2000) Chem Eng News 78:41).

10. Methods and assays for identifying any form of nucleic acid binding a) Competition Dialysis of Neomycin-Acridine Conjugate with Nucleic Acid Forms

237. In addition to stabilizing DNA, RNA, and hybrid triple helices, neomycin also induces the stabilization of hybrid duplexes as well as hybrid triple helices (Arya DP, et al (2001) J Am Chem Soc 123:11093). This significantly adds to the number of nucleic acids (other than RNA) that aminoglycosides can target. A rapid technique has been established for a quantitative assay to determine the relative binding affinities for host triplex, duplex DNA, singlestranded (SS) DNA/RNA and other possible nucleic acid targets (tetraplex) for a given

aminoglycoside ligand using a thermodynamically rigorous competitive equilibrium dialysis method that exploits therapeutically useful drug concentrations (Ren J, et al (2000) J Am Chem Soc 122:424; Ren J, et al (2001) Methods Enzymol, vol 34O.Academic,New York, p 99). In the assay, solutions consisting of identical concentrations of different nucleic acid structures were dialysed simultaneously against a common ligand dissolved in appropriately buffered conditions. After equilibration, the amount of ligand bound to each DNA was measured by spectrophotometry. More ligand accumulated in the dialysis tube containing the structural form of highest binding affinity and, since all of the DNA samples were in equilibrium with the same free ligand concentration, the amount of ligand bound was directly proportional to the binding constant for each con-formational form. Thus, comparison among the DNA samples gave a rapid and thermodynamically reliable indication of structural selectivity for any given ligand.

238. Since aminoglycosides do not have a chromophore for spectrophotometric analysis, competition dialysis of three acridines with increasing positive charge was used to decipher aminoglycoside specificity (Figure 11). Competition dialysis studies were carried out using 9-aminoacridine, quinacrine, and a neomycin-acridine (neo-acridine) conjugate (Kirk SR, et al (2000) J Am Chem Soc 122:980) against 14 different nucleic acids. Going from acridine to neo-acridine, the effect of neomycin conjugated to the acridine chromophore was evaluated. At ^• first sight, dialysis of neo-acridine (Figure 12) showed highly promiscuous binding with little preference for any specific nucleic acid structure, except for a clear preference for RNA triplex. Among comparable single strand, duplex, and triplex structures, maximum binding was always observed with the triplexes. This seemingly promiscuous binding yielded a different picture upon careful analysis of the dialysis data. All three drugs showed comparable binding to one nucleic acid: calf thymus DNA. Calf thymus DNA also represents a standard duplex DNA. This observation was used to replot the dialysis results to emphasize differences relative to that standard. These results, shown in Figure 13, better illustrate the change in specificity of the different acridines toward different nucleic acids. While 9-aminoacridine and quinacrine showed a clear preference for DNA triplex, neo-acridine binding to RNA triplex is much greater than DNA triplex and even better than the natural aminoglycoside RNA target: eubacterial 16S A- site. Drug binding was also observed with DNA as well as RNA duplex, and even with DNA tetraplex. The binding to DNA tetraplex was still lower than to the RNA triplex. RNA»DNA duplexes were better targets than DNA homoduplexes; poly(dA)»poly(rU) hybrid duplex being comparable in binding to the tetraplexes. Also observed was the significant binding with the poly(dG)»poly(dC) duplex.

2 ^" 39 ^" A competition 'dialysis assay using tenfold (100 riM) and 100-fold (10 nM) lower concentrations (nanomolar range) was also carried out. Results from dialysis under 100 nM drug concentration (Figure 14) showed that neo-acridine favors nucleic acid forms that can adopt an A-type conformation. However, reliable results could not be obtained at 1 nM and 10 nM concentrations due to the low fluorescence intensity of the neo-acridine conjugate.

240. Neo-acridine binding to RNA triplex was also investigated by UV thermal melts, ITC, viscometric and CD titrations. Thermal denaturation in the presence of neo-acridine showed an increase in T _{m3→ 2} at low drug concentrations. At higher drug concentrations, the duplex was stabilized as well. Neomycin is one of the best stabilizers, of an RNA triple helix (Arya DP, et al (2001) J Am Chem Soc 123:538.5). Viscosity measurements showed a clear groove binding (as seen by shortening of RNA triplex length) upon titration of neomycin as well as neoacridine into the triplex (Arya DP, et al (2003) J Am Chem Soc 125:10148).

11. Forms of nucleic acids a) Propensity Towards an A-type Conformation

241. RNA duplex structures are known to adopt an A-type conformation, as are hybrid duplexes (Sanger W (1983) In: Cantor CR (ed) Principles of nucleic acid structure. Springer, Berlin Heidelberg New York, p 242). dG.dC-rich DNA duplex sequences (Stefl R, et al J (2001) J MoI Biol 307:513) have also been shown to have a high propensity for A-form in the presence of cations, including neomycin (Robinson H, et al (1996) Nucleic Acids. Res 24:676), and CD studies have suggested the A-like solution conformation of G4 tetraplexes (KyprJ, et al (2001) Eurbiophysi 30:555). Further evidence of A-type preference was observed with the change in the CD spectrum of poly(dG)»poly(dC) upon inclusion of neo-acridine. A shift in λmaxfrom 257 nm to 267 nm, and increased signal in this range, in the presence of this drug, was strongly indicative ofaB→ A transformation (Robinson H, et al (1996) Nucleic Acids Res 24:676; Stefl R, et al J (2001) J MoI Biol 307:513; KyprJ, et al (2001) Eurbiophysi 30:555). Additionally, the differences in binding to DNA»RNA hybrids can be attributed to the fact that poly(dA)«poly(rU) has been known to adopt an A-type conformation whereas poly(rA)»poly(dT) can exist in the B- form (Sanger W (1983) hi: Cantor CR (ed) Principles of nucleic acid structure. Springer, Berlin Heidelberg New York, p 242). b) Importance of A-Form DNA and its Recognition

242. The polymorphism of DNA was noticed early after the discovery of its double helical structure (Franklin RE, et al (1953) Acta Crystallography 6:673). The conformations of DNA have since been limited to two major distinctions: A-DNA and B-DNA (other less-well-

known structures 35 exist). Both structures are of identical topology and hydrogen bonding patterns, but they differ largely in their overall shape (Figure 15). B-DNA has long been believed to be the dominant biological conformation, implementing water molecules and biological cations appropriately within its structure. A-DNA, on the other hand, requires dehydrated conditions. The transition of B- to A-DNA is a reversible and cooperative process (Lvanov VI, et al (1995) Molekulyamaya Biologiya (Moscow) 28:780), in which the A-form is considered the higher-energy state. The underlying factors for this instability have been addressed, but with little success (Calladine CR, et al (1984) J MoI Biol 178:773; Marky NU, et al (1994) Biopolymers 34:121).

243. Native DNA, which comprises the genetic information of all known free organisms, mostly adopts B-form under physiological conditions because it is associated with high humidity in fibers or with aqueous solutions of DNA. However, it is important to switch B- DNA into the A-form in a living organism since constitutive conformation of double-stranded RNA is predominantly A-type. RNA probably preceded DNA in evolution (Jeffares DC, et al (1998) J MoI Evol 46:18), so the basic mechanisms of genetic information copying are likely to have evolved on an A-form rather than B-form. hi fact, the template DNA is induced by many polymerases into A-form at positions of genetic information copying in the microenvironment. Thus, DNA switching into A-form can influence replication and transcription of the genomes.

244. Understanding aminoglycoside A-form nucleic acid interactions then has underlying importance to the area of drug development as well as to the fundamental understanding of A-form recognition because: 1. nucleic acid therapeutic targets can be identified with a better understanding of the thermodynamics and kinetics of molecular recognition involved in aminoglycoside specificity; 2 as opposed to B-form DNA recognition, very few small molecules (multivalent cations) (Lvanov VI, et al (1995) Molekulyamaya Biologiya (Moscow) 28:780; Lu X-J, et al (2000) J MoI Biol 300:819; Robinson H, et al (2000) Nucleic Acids Res 28:1760) are known that select for A-form structural features. Aminoglycosides present a novel scaffold for groove recognition of A-form structures; 3. Aminoglycoside binding to such higher order structures (H-DNA triplex) has also been implicated in their toxic side effects (Arya DP, et al (2001) J Am Chem Soc 123:5385). A better understanding of aminoglycoside binding and selectivity can also help in a better understanding of toxic side-effects of these broad spectrum antibiotics. c) From A- to B-Form Nucleic Acids: Using Organic Chemistry to Tune Aminoglycoside Selectivity

245. Aminoglycosides bind in the major groove of A-form structures (much like RNA, as the A-form nucleic acids have a narrower major groove) (Arya DP, et al (2003) J Am Chem Soc 125: 10148). The B-form duplex has a much larger major groove and does not provide a good shape complementarity for aminoglycoside binding (see Figure 16). These findings have complemented the success in development of DNA duplex-specific groove binders in the past few decades, among which netropsin, distamycin and Hoechst 33258 have been the lead compounds. Conjugation of neomycin to Hoechst 33258 was accomplished to investigate whether a molecule like neomycin could be forced into the B-form DNA major groove and to determine if binding would be driven by Hoechst 33258 (duplex-selective groove binder) or neomycin (triplex-selective groove binder). Such ligands with minor/major groove recognition are promising drug candidates for development of inhibitors of transcription factors (White CM, et al (2001) Proc Natl Acad Sci USA 98:10590).

246. An exemplary synthesis of neomycin to Hoechst 33258 is shown in Figure 17. Starting from the natural product neomycin B, which is commercially available as the tri-sulfate salt, Boc (t-butoxycarbonyl) protection of the six amino groups followed by conversion to 2,4,6- triisopropylbenzenesulfonyl derivative, and subsequent substitution by aminoethanethiol, gave rise to the protected neomycin amine (Kirk SR, et al (2000) J Am Chem Soc 122:980) compound 4. Treatment of 4 with l,l'-thiocarbonyldi- 2(lH)-pyridone using a catalytic amount of DMAP gave isothiocyanate derivative 5, which was coupled with bis(benzimidazole) 3 and deprotected to give conjugate 7 (Figure 17).

247. The thermal stability of DNA triple and double helices in the presence of neomycin, Hoechst 33258, and neomycin-Hoechst 33258 conjugate 7 was investigated using thermal denaturation monitored by UV absorbance. It was found that 7 displayed a marked effect on the stability of poly(dA)»poly(dT) duplex when compared to both neomycin (which is known to have no effect on the thermal stability of duplex DNA) and Hoechst 33258,which displayed some degree of stabilization of duplex DNA (Figure 18).

248. In the absence of ligand, the melting profile of poly(dA)»2poly(dT) is biphasic with T _{m3→ 2} =34°C and T _m2→ ^72 ⁰ C. As depicted in Figure 18, the dissociation of duplex DNA in the presence of 7 occurs at a higher temperature (>95°C) than that of DNA in the presence of Hoechst 33258 (86 ⁰ C) and neomycin (72 ⁰ C, unchanged when compared to native duplex melting). This suggests that 7 stabilizes the duplex better than the individual parent compounds. Samples containing both neomycin and Hoechst 33258 displayed no difference in T _m from that observed with the individual molecules. It is important to note that triplex melting was not

dbserve3 ^" for pbly(dA ^~ )»2p ^' ory(dT) in the presence of 7, suggesting that drug binding prevents the third strand polypyrimidine from binding in the major groove. A comparison was then made with a self-complementary DNA duplex d(CGCAAATTTGCG) ₂ well known for Hoechst 33258 affinity (Haq I, et al (1997) J MoI Biol 271:244). UV melting showed increased stability of the duplex in the presence of 7, with a δT _m =25°C, compared to δT _m =14°C for Hoechst 33258 (Haq I, et al (1997) J MoI Biol 271 :244).

249. Further studies of numerous duplex DNA 22-mers of varying G/C content (breaking up stretches of A/T base pairs) were carried out. In all cases where stretches of at least 4 base pairs were present, λT _m for 7 was at least 10 ⁰ C higher than that for Hoechst 33258. Duplex stabilization by 7 followed the selectivity shown by Hoechst 33258 (Figure 19a), whereas neomycin had no effect on the stabilization of any duplex. Hoechst 33258 is well known to have a primary preference for A/T stretches as low as four base pairs, suggesting that the binding- induced thermal stabilization by 7 is largely controlled by the Hoechst 33258 moiety's ability to bind to its required stretch of A/T base pairs. A model depicting the possible binding of 7 to a 12-mer duplex is shown in Figure 19b. Computer modeling suggests that electrostatic and H-bonding contacts between neomycin and sites within the major groove compete somewhat with the otherwise deep minor groove binding of Hoechst 33258 (Figure 19b). As Hoechst 33258 binds in the minor groove, neomycin is unable to be completely buried in the major groove (due to the linker size). Despite this constraint, conjugate 7 prefers the duplex, suggesting that neomycin can be forced into the major groove of a B-form DNA duplex. In retrospect, this could be due primarily to the larger binding constants observed between Hoechst 33258 and duplex DNA (Haq I, et al (1997) J MoI Biol 271 :244) (~10 ⁸ M ^"1 ) as opposed to neomycin binding to triplex (10 ⁵ -10 ⁶ M ^~1 )(AryaDP, et al (2003) J Am Chem Soc 125:3733). Conjugates of different linker sizes can then be designed to target a structure of preference and can aid in the development of even more selective and potent conjugates.

12. Targeting Nucleic Acids with Aminoglycoside-DNA and PNA Conjugates a) RNA Sequence-Specific Aminoglycoside-ODN Conjugates

250. KNA has now become a well-established drug target (Hermann T, et al (1998) Curr Opin Biotechnol 9:66; Ecker DJ, et al (1999) Drug Discovery Today 4:420; Gallego JVU (2001) Ace Chem Res 34:836). Small molecules and antisense oligonucleotides are now being used to down-regulate gene expression. Vitravene, the first antisense drug, was approved by the FDA at the end of the 20th century (Grillone LR (2001) hi: Crooke ST (ed) Antisense drug technology: principles, strategies and applications. Dekker,New York, p 725). RNA has distinct

advantages ^" m ^" antibacterial arid antiviral treatment. Primarily, appearance of drug resistance through point mutations in a conserved RNA motif among bacteria or viral strains is likely to be slow. Bacteria become resistant to ribosomal RNA-binding antibiotics through exchange of genetic material encoding RNA-modifying enzymes (typically methyltransferases and phosphotransferases), drug-modifying enzymes, or enzymes that affect drug transport (Neu HC (1992) Science 257:1064; Azucena E, et al (2001) Drug Resist Update 4:106). Therefore, if the structure of the nucleic acid-binding drug is novel, the emergence of resistance is likely to be slower than for protein targets (barring any novel efflux pump mechanisms). Antisense/antigene therapy can offer a viable alternative in tackling such resistance mechanisms.

251. As provided herein, conjugation of an aminoglycoside to an ODN can assist in the following processes: 1. Delivery of aminoglycoside to a specific DNA/RNA site; 2. Increasing the stabilization inferred by these hybrid duplex/triplex stabilizing agents; 3. The unique structure of aminoglycosides can aid in cellular permeability/ site-specific delivery of the ODN

252. To investigate the advantage of nucleic acid-based specificity coupled with aminoglycoside charge/shape complementarity, provided is a general strategy for the synthesis of covalently attaching aminoglycosides (neomycin) to nucleic acid analogs (DNA/PNA). Ultrarapid functional genomics technologies have helped identify approximately 4,000 essential gene drug targets in 11 clinically relevant bacterial and fungal pathogens (Davies J, et al (1968) J Biol Chem 243:3312; Elitra (1999) Bioworld Week 7:4; Haselbeck R, et al (2002) Cur Pharm Des 8:1155). In contrast, most antimicrobials prescribed today inhibit only a small fraction of this number of targets within bacterial and fungal pathogens. A comprehensive approach to identifying such essential drug targets in multiple pathogens can be combined with a complementary approach of developing antimicrobial agents that are sequence-specific to previously known, as well as rapidly identified, new RNA targets. Interestingly, a shotgun antisense technology was used as the key tool to identify these 4,000 essential genes, suggesting that oligos binding to these RNA targets will be able to selectively shut down protein synthesis. Additionally, aminoglycosides (neomycin)-ODN conjugates can also be effective models for targeting nucleic acids sequence-specifically via triplex or hybrid duplex formation. b) Synthesis of Aminoglycoside Isothiocyanates/ODN- Aminoglycoside Conjugate

253. The amino groups on rings I, II, and IV (neomycin) are necessary in recognizing and in stabilizing various nucleic acid forms (aminoglycosides without any of these amines do not stabilize DNA triplexes as efficiently) (Charles I, et al (2002) Bioorg Med Chem Lett

12:1259). The conjugates based on aminoglycosides must then retain these amines. The 5"-OH on ring HI (neomycin) was chosen to provide the linkage to the nucleic acids (for ring numbering, please see Figure 1). The synthesis of neomycin isothiocyanate has been reported as a stable reagent that can be coupled to a variety of amines (Charles I, et al (2002) Bioorg Med Chem Lett 12:1259, herein incorporated by reference for the teaching of neomycin coupling to amines). Figure 17 shows the synthesis of neomycin isothiocyanate, starting from neomycin amine. The use of this isothiocyanate in the synthesis of a DNA 5'— aminothymidine dimer conjugated to neomycin and kanamycin also has been recently reported (Figure 20) (Charles I, et al (2002) Bioorg Med Chem Lett 12:1259). c) Synthesis of Oligomeric Neomycin-ODN Coniugates (1) Neomycin-DNA/PNA Conjugate

254. The structures of generic neomycin DNA and PNA conjugates is shown in Figure 21a (Charles I, et al (2004) Bioorg Med Chem Lett (in press)). The synthesis of neomycin conjugated to 5'-end of a oligonucleotide dT ₍₁₆₎ is shown in Figure 21b. Neomycin is linked to the DNA via a thiourea linkage. Neomycin isothiocyanate (Figure 21b) has been coupled to a 5'- amino-5'-deoxy ODN, which is easily prepared by incorporation of 5'-amino-5' deoxythymidine (or cytidine) in a growing ODN chain. The synthesis of neomycin linked to a 16-mer DNA dT ₍₁₆ ) has been reported (Charles, I.; Arya, D. P. J. Carbohydr. Chem. 2005, 24, 143-157. ). The reactive amine at the 5'-end of dT ₍₁₆₎ (Figure 21b) was treated with a pyridine solution containing neomycin isothiocyanate and 4-dimethylaminopyridine (DMAP) for 12 h at room temperature, washed with trifluoroacetic acid (TFA), and deprotected from solid support with NH ₄ OH (Charles I, et al (2004) Bioorg Med Chem Lett (in press)). Having established that these conjugates can be synthesized on solid phase using conventional DNA synthesis, attention can now be devoted to synthesizing ODNs for targeting anticancer and antimicrobial DNA sequences of interest (Charles I, et al (2004) Bioorg Med Chem Lett (in press)). (Vazquez ICM, et al (2002) Q Rev Biophys 35:89).

255. As provided herein, aminoglycosides can considerably enhance the binding affinities of the ODNs to their duplex DNA target as well as to the single strand RNA targets. This approach can open up doors for developing sequence-specific anticancer and antimicrobial drugs.

13. AT-rich

256. "Control region" refers to specific sequences at the 5' and 3' ends of eukaryotic genes which maybe involved in the control of either transcription or translation. Virtually all

eukaryotic genes have an AT-πch region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CXCAAT region where X may be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence which may be the signal for addition of the poly A tail to the 3' end of the transcribed mRNA. 14. Exemplary targets and methods

257. It is understood that the B-form binders can be used to target and bind any B-form double stranded helix as described herein. The A-form binders can be used to target and bind any A-form double stranded helix as described herein. The single stranded binders can be used to target and bind any single stranded target sequence. The use of the disclosed compositions for binding can be performed as disclosed herein, but can include, for example, administration, in vitro, in vivo, ex vivo, to a cell, to an organism, such as an animal, such as a mammal, such as a human, chimpanzee, ape, cat, dog, rabbit, mouse, or rat, for example. The administration can be as described herein and can include, for example, delivery via liposomes or other vehicles. The method can involve bringing into contact one or more of the disclosed compositions for binding with one or more cells, introducing one or more of the disclosed compositions for binding into one or more cells, and/or mixing one or more of the disclosed compositions for binding with one or more cells. Such modes can be considered forms of administration. The cells can be, for example, individual cells, cells in tissues, cells in culture, and/or cells in or out of organisms. The adminitered compositions can bind to the target nucleic acid in the cells and have an effect, which can depend on the target nucleic acid, the target sequence, and/or the normal, natural or current role or function of the target nucleic acid and the form of the composition for binding that is used.

258. The disclosed compositions for binding can have any of a variety of effects, which can depend on the target nucleic acid, the target sequence, and/or the normal, natural or current role or function of the target nucleic acid and the form of the composition for binding that is used. For example, it is understood that the disclosed compositions and administration can be used to inhibit or prevent protein-nucleic acid interactions, nucleic acid-nucleic acid interactions, cellular replication or differentiation, bacterial or viral life cycles, or other pathogen life cycles. The disclosed compositions and method can be used to alter or affect the function, regulation and/or effect of the target nucleic acid. For example, it is understood that the disclosed compositions and method can be used to target specific genes, DNA, and regions of, for example, ribosomal or viral RNA, or messenger RNA, as disclosed herein. It is also

understood that gene expression and or regulation can be inhibited, altered, manipulated, and/or disrupted. It is understood that the disclosed compositions can be administered to any organism for treatment of a particular disorder, which is related to the composition binding the nucleic acid target. It is understood that the disclosed compositions can be used for, for example, inhibition, alteration, manipulation, and/or disruption of replication, transcription, translation, translation machinery, and/or tRNA binding.

259. It is also understood that the disclosed compositions can be used for marking or labeling a particular nucleic acid sequence in vitro, in vivo, or ex vivo. Such binding can be used on, for example, cell-free, recombinant, in vitro synthesized, purified and/or extracted nucleic acid, and/or nucleic acid in any cell (cells in vitro, cell ex vivo, cells in vivo, cells in tissue, cells in organism). Every aspect that can be used for administration for binding a particular target sequence which is capable of, for example, treating a particular disorder, such as a baterial infection, can also be administered for labeling or diagnmostic purposes as well. a) Exemplary Ribsomal RNA targets

260. Figures 22-23 shows 16s ribosomal RNA sequences that can be targeted by the disclosed compositions, and thus the disclosed compositions can be used as antibiotics. The double stranded regions can be targeted, for example, by the A-form binders as disclosed herein.

In addition, each single stranded region can be targeted with a single stranded nucleic acid binder as described herein, such as a complementary ODN with an aminoglycoside conjugated to it. It is also understood that the disclosed compositions where, for example, an A-form binder and a single stranded nucleic acid binder are conjugated and used to target a region which has single stranded RNA, for example, contiguous with a double stranded region can be used for targeting the appropriate regions. Each and every single stranded region shown in figures 24-26 is a potential target, partially or wholly, and thus each and every complementary ODN is disclosed herein for these regions. Likewise, each double stranded region is a target for the appropriate disclosed compositions as well. b) Exemplary RNA viral targets

261. Figure 24-26 show the packaging RNA, RRE and TAR regions of HW respectively as exemplary RNA virus targets. The double stranded regions can be targeted, for example, by the A-form binders as disclosed herein. In addition, each single stranded region can be targeted with a single stranded nucleic acid binder as described herein, such as a complementary ODN with an aminoglycoside conjugated to it. It is also understood that the disclosed compositions where, for example, an A-form binder and a single stranded nucleic acid

binder are conjugated arid used to target a region which has single stranded RNA, for example, contiguous with a double stranded region can be used for targeting the appropriate regions. Each and every single stranded region shown in figures 24-26 is a potential target, partially or wholly, and thus each and every complementary ODN is disclosed herein for these regions. Likewise, each double stranded region is a target for the appropriate disclosed compositions as well. c) Exemplary gene and mRNA targets

(1) c-myc

262. Available evidence suggests that a family of tumors, including Burkitt's lymphoma and others, share a common genetic lesion, which is evident as constitutive overproduction of the c-myc mRNA and its corresponding c-myc protein. Because the c-myc protein has been shown to be a critical element in the control of cell growth, it is believed that there may be a direct causal relation between the overproduction of c-myc protein and uncontrolled cancerous growth for such cells. .

263. In both cancerous and normal cells, the c-myc gene possesses several target sequences within its 5.' flanking sequence which satisfy the synthetic oligonucleotide design criteria, hi a program of drug development, these target sequences and others are used as templates to direct oligonucleotide design. The purpose of these oligonucleotides is to selectively inhibit c-myc transcription, thereby repressing the uncontrolled growth of tumors with the c-myc lesion. Triplex forming oligonucleotides (TFO) have been used to inhibit transcription of c-myc (see U.S. Patent 5,176,996, herein incorporated by reference in its entirety for its teaching of c- myc TFOs).

264. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -153/-116 upstream of the c-myc gene translation initiation point. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -142/-115 upstream of the c-myc gene translation initiation point. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -61 /-16 upstream of the c-myc gene translation initiation point. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -87/-58 upstream of the c-myc gene translation initiation point. The provided oligonucleotide can comprise the nucleic acid sequence SEQ ID NO:1, 2, 3, 4, 5, 6, 7, or 8.

(2) Collagen I

265. The structural proteins which define the mechanical properties of skin are well known. The molecular structure of the collagen and elastin proteins and their corresponding

proteases, ^{' "} cdllagenase arid eTastase, have been intensley studied. These proteins are under the control of an elaborate program of regulation, which appears to change during the wound healing process and as a result of the aging process. The molecular structure is sufficiently defined to consider treatments based upon gene-specific intervention into the pattern of structural protein synthesis and/or enzymatic degradation.

266. Data suggest that the change in the mechanical properties of skin which accompanies aging (wrinkling, etc.) is due in part to an age-specific change in the relative abundance of the collagens and other structural proteins. Interference with the synthesis and/or selective degradation of these proteins by drug treatment can reestablish a distribution which approximates that of younger tissue, and thus the effects of aging can be partially reversed.

267. A program of synthetic oligonucleotide design, based upon manipulation of collagen I synthesis in human skin is described below. By altering the relative protein concentrations the structure and mechanical properties of skin can be altered. Thus the synthetic oligonucleotide can be used as a therapeutic agent to alter the skin aging process or to alter the wound healing process. One skilled in the art will readily recognize that the concepts can be extended to other collagens, to other skin proteins and to their complementary proteases based upon the availability of the necessary genetic data.

268. Representative target sequences in the transcription control region of the human αl(I) collagen gene, the likely function of those sites, their position relative to the RNA transcription origin, and the synthetic oligonucleotide sequence designed for collagen specific treatment as shown below. As the molecular genetics of the collagen gene develops, the list of target sequences within the 5' flanking region will be expanded. Triplex forming oligonucleotides (TFO) have been used to inhibit transcription of collagen/collagenase (see U.S. Patent 5,176,996, herein incorporated by reference in its entirety for its teaching of collagen/collagenase TFOs).

269. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -168/-124 upstream of the human αl(I) collagen gene translation initiation point. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -294/-264 upstream of the human αl(I) collagen gene translation initiation point. The provided oligonucleotide can comprise the nucleic acid sequence SEQ ID NO:84, 85, 86, or 87.

270. Also provided are oligonucleotides that inhibit collagenase protein synthesis. The process includes specific repression of collagenase RNA transcription. The oligonucleotide an causes loss of TPA sensitivity, and a subsequent repression of collagenase syntheses in the

pfe ^' serice ^' ofpfomoitors such as TPA. This process includes specific repression of collagenase RNA transcription. Synthetic oligonucleotide interaction can cause collagen protein levels in the cell to rise, as collagenase levels fall. The clinical effect of the increase can cause a useful alteration of the mechanical properties of skin. The effects can be seen by adding sufficient amounts of oligonucleotide for cellular uptake to cultured human fibroblasts.

271. Thus, the provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -48/- 16 upstream of the collagenase gene translation initiation point. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -91 /-64 upstream of the collagenase gene translation initiation point. The provided oligonucleotide can comprise the nucleic acid sequence SEQ ID NO:9, 10, 11, or 12.

272. One skilled in the art will readily appreciate that these concepts can be extended to other genes which are known to be involved in skin development, repair and aging and is only limited by the available molecular genetic data.

(3) HIV

273. The HTV-I virus is known to be the causative agent in human acquired immune deficiency syndrome (ADDS). The long terminal repeat of the HIV-I virus is known to possess several DNA segments within the LTR region which are required for transcription initiation in a human T-cell host. The synthetic oligonucleotides selectively repress HIV-I mRNA synthesis in a human host cell, by means of triplex formation upon target sequences within the viral LTR. Repression of an RNA synthesis results in the reduction of the growth rate of the virus. This could result in the slowing of the infection process or the repression of the transition from latency to virulent growth. Most of the sites within the LTR will comprise target sites for drug (oligonucleotide) intervention. There is no wasted DNA in the small, highly conserved LTR region.

274. Triplex forming oligonucleotides (TFO) have been used to inhibit transcription of HIV-I LTR (see U.S. Patent 5,176,996, herein incorporated by reference in its entirety for its teaching of HIV TFOs).

275. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -80/-51 upstream of the HIV-I gene translation initiation point. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -16/+13upstream of the HIV- 1 gene translation initiation point. The provided oligonucleotide can comprise the nucleic acid sequence SEQ DD NO: 13, 14, 15, or 16.

(4) APP770

276. The APP770 ^" Gene is the precursor protein responsible for production of plaque in Alzheimers disease. Triplex forming oligonucleotides (TFO) have been used to inhibit transcription of APP770 (see U.S. Patent 5,176,996, herein incorporated by reference in its entirety for its teaching of HIV TFOs).

277. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -1121-619 upstream of the APP770 gene translation initiation point. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -618/-590 upstream of the APP770 gene translation initiation point. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -477/-440 upstream of the APP770 gene translation initiation point. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -434/-407 upstream of the APP770 gene translation initiation point. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -286A252 upstream of the APP770 gene translation initiation point. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -264/-230 upstream of the APP770 gene translation initiation point. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -200/- 177 upstream of the APP770 gene translation initiation point. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -40/-9 upstream of the APP770 gene translation initiation point. The provided oligonucleotide can comprise the nucleic acid sequence SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32..

(5) EGFR

278. Inappropriately high expression of the epidermal growth factor gene (EGFR) has been implicated as crucial to the development of cancers and several skin diseases (psoriasis). Triplex forming oligonucleotides (TFO) have been used to inhibit transcription of EGFR (see U.S. Patent 5,176,996, herein incorporated by reference in its entirety for its teaching of HTV TFOs).

279. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -109/-83 upstream of the EGFR gene translation initiation point. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -307/-281 upstream of the EGFR gene translation initiation point. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -352/-317 upstream of the EGFR gene translation initiation point. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -363/-338 upstream of the EGFR gene translation initiation point. The

pfovided ^'" δlϊgδnucrebtϊde''cari'"βomprise the nucleic acid sequence SEQ ID NO:33, 34, 35, 36, 37, 38, 39, or 40.

(6) GSTpi

280. Overexpression of the enzyme gluththione-s-transferase pi (GSTpi) has been implicated as being responsible for the broad-range drug resistance which develops in a variety of cancers. The synthetic oligonucleotides described below are designed to repress GST-pi expression, thereby sensitizing cancerous tissue to traditional drug chemotherapy. The target domain can comprise the consensus binding sequences for the transcription activating factors API and SpI. Synthetic Oligonucleotides targeted against this will repress GSTpi transcription by means of competition with API and SpI. Triplex forming oligonucleotides (TFO) have been used to inhibit transcription of GSTpi (see U.S. Patent 5,176,996, herein incorporated by reference in its entirety for its teaching of HIV TFOs).

281. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -68A39 upstream of the GSTpi gene translation initiation point. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -227/-204 upstream of the GSTpi gene translation initiation point. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -499/-410 upstream of the GSTpi gene translation initiation point. The provided oligonucleotide can comprise the nucleic acid sequence SEQ ID NO:41, 42, 43, 44, or 45.

(7) HMGCoA Reductase

282. HMGCoA Reductase is the enzyme which defines the rate limiting step in cholesterol biosynthesis. Its molecular genetics has been studied to understand the control of cholesterol synthesis. The described synthetic oligonucleotides will intervene in the program of cholesterol synthesis by means of modulating the transcription of HMGCoA. Triplex forming oligonucleotides (TFO) have been used to inhibit transcription of HMGCoA Reductase (see U.S. Patent 5,176,996, herein incorporated by reference in its entirety for its teaching of HIV TFOs).

283. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -167/-135 upstream of the HMGCoA Reductase gene translation initiation point. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -134/-104 upstream of the HMGCoA Reductase gene translation initiation point. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -41 /-6 upstream of the HMGCoA Reductase gene translation initiation point. The provided oligonucleotide can comprise the nucleic acid sequence SEQ ID NO:46, 47, 48, 49, 50, or 51.

(8) NGFR

284. The NGFR gene encodes a cell surface receptor required for nerve cell proliferation. It is overexpressed in neuroblastoma and melanomas. Triplex oligonucleotides can be designed to repress the growth of those cancerous tissues. Activation of the gene would be a precondition of activation of nerve cell regeneration. Triplex forming oligonucleotides (TFO) have been used to inhibit transcription of NGFR (see U.S. Patent 5,176,996, herein incorporated by reference in its entirety for its teaching of HTV TFOs).

285. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -323/-290 upstream of the NGFR gene translation initiation point. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -309/-275 upstream of the NGFR gene translation initiation point. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -2S5/-248 upstream of the NGFR gene translation initiation point. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -243/-216 upstream of the NGFR gene translation initiation point. The provided oligonucleotide can comprise the nucleic acid sequence SEQ ID NO: 52, 53, 54, 55, 56, 57, 58, or 59.

(9) HSV-I Polymerase

286. HSV-I is responsible for a variety of skin lesions and other infections. The triplex oligonucleotide can be designed to bind directly to the promoter region of the genes which encode the viral DNA polymerase and DNA binding protein, thereby arresting viral replication. Triplex forming oligonucleotides (TFO) have been used to inhibit transcription of HSV-I promoter (see U.S. Patent 5,176,996, herein incorporated by reference in its entirety for its teaching of HIV TFOs).

287. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -60/-26 upstream of the HSV-I polymerase promoter gene translation initiation point. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -82/- 118 upstream of the HSV-I polymerase promoter gene translation initiation point. The provided oligonucleotide can comprise the nucleic acid sequence SEQ ID NO:60, 61, 62, or 63.

(10) HSV-I origin of replication

288. The triplex oligonucleotides can be designed to bind directly to the two classes of HSV-I DNA replication origin, thereby arresting viral replication. The first origin (oriL) occurs at 0.4 map units and is in between and immediately adjacent to the HSV-I DNA polymerase and DNA binding protein genes. The two identical origins of the second type (oriS) occur at 0.82 and

0.97 map ^" unϊis ^' . ^' Numbering below is the terms of position relative to the two fold symmetry axis of each origin. Triplex forming oligonucleotides (TFO) have been used to inhibit transcription of HSV-I origin of replication (see U.S. Patent 5,176,996, herein incorporated by reference in its entirety for its teaching of HTV TFOs).

289. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -48/- 10 relative to the two fold symmetry axis of HSV-I origin. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region +10/+47 relative to the two fold symmetry axis of HSV-I origin. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region +69λ34 relative to the two fold symmetry axis of HSV-I origin. The provided oligonucleotide can comprise the nucleic acid sequence SEQ ID NO:64, 65, 66 or 67.

(11) Beta Globulin

290. The beta globin gene encodes one of the proteins comprising adult hemoglobin. Mutation in this gene is responsible for beta thalassemia and sickle cell anemia. Triplex oligonucleotides targeted to this gene are designed to inhibit the beta globin gene in thallassemics and in patients with sickle cell anemia, to be replaced by the naturally occurring delta protein. Two classes of triplex oligonucleotides TFO are described, which are targeted against the 5' enhancer or the promoter/coding domain. Numbering is relative to the principal mRNA start site. Triplex forming oligonucleotides (TFO) have been used to inhibit transcription of beta globin (see U.S. Patent 5,176,996, herein incorporated by reference in its entirety for its teaching of HTV TFOs).

291. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -912/-886 upstream of the beta globin gene translation initiation point. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -63A25 upstream of the beta globin gene translation initiation point. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -361-9 upstream of the beta globin gene translation initiation point. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region +514/+543 upstream of the beta globin gene translation initiation point. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region +693/+719 upstream of the beta globin gene translation initiation point. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region +874/+900 upstream of the beta globin gene translation initiation point. The provided oligonucleotide can comprise the nucleic acid sequence SEQ ID NO:68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, or 79.

<<:

(12) IL15

292. Interleukin-15 is a novel cytokine having biological functions similar to those of interleukin-2 even though there is no significant sequence homology between the two. Interleukin-15 is produced by epithelial and fibroblast cell lines, and by peripheral blood monocytes. Furthermore, interleukin-15 -specific rnRNA has been found in several normal human tissues including placenta, skeletal muscle, and kidney (Grabstein et al., 1994, Science 264:965-968).

293. Merleukin- 15 (IL- 15) induces T-cell proliferation, enhances natural killer (NK) cell cytotoxicity and antibody-dependent cell-mediated cytotoxicity, and upregulates production of NK cell-derived cytokines including interferon-γ. (IFN- γ), granulocyte/macrophage-colony- stimulating factor (GM-CSF), and tumor necrosis factor-α (TNF-α) (Grabstein et al., 1994, Science 264:965-968; Burton et al., 1994, Proc. Natl. Acad. Sci. 91:4935-4939; Bamford et al.,

1994, Proc. Natl. Acad. Sci. 91:4940-4944; Giri et al., 1994, EMBO J. 13:2822-2830; Carson et al., 1994, J. Exp. Med. 180:1395-1403; and Giri et al., 1995, EMBO J. 14:3654-3663). IL-15 also co-stimulates proliferation and differentiation of B cells activated with antiimmunoglobulin M (anti-lgM) (Armitage et al., 1995, J. Immunol. 154:483-490), stimulates locomotion and chemotaxis of normal T cells (Wilkinson et al., 1995, J. Exp. Med. 181:1255-1259), and promotes interleukin-5 production by T cells which may contribute to eosinophilic inflammation (Mori et al., 1996, J. Immunol. 156:2400-2405). Persistant eosinophilic inflammation in the bronchial mucosa is well recognized in the pathogenesis of chronic asthma (Bousquet et al., 1990, N. Engl. J. Med. 323:1033).

. 294. Rheumatoid arthritis is a destructive inflammatory polyarthropathy (Maini et al.,

1995, in Mechanisms and Models in Rheumatoid Arthritis, pp. 25-26, eds. Henderson, Edwards, and Pettifer, Academic Press, London, 25-26). Chronic rheumatoid synovitis is characterized by the presence of activated fibroblast-like synoviocytes together with infiltration of the normally acellular synovial membrane by macrophages, T cells, and plasma cells (Duke et al., 1982, Clin. Exp. Immunol. 49:22-30). Levels of IL-15 in rheumatoid arthritis synovial fluid are sufficient to exert chemoattractant activity on T cells in vitro, and can induce proliferation of peripheral blood and synovial T cells; furthermore, IL-15 induces an inflammatory infiltrate consisting predominantly of T lymphocytes (Mclnnes et al., 1996, Nature Medicine 2:175-182). Therapies directed at T cells, such as cyclosporin A and monoclonal antibodies against T-cell surface antigens, produce significant clinical improvement, confirming the importance of T cells in inflammatory polyarthropathy (Homeff et al., 1991, Arth. Rheum. 34:129-140; Wendling et al.,

l ^' 9 ^' 9l7X ^"' KEeύmatoL l8 ^" :325-3 ^' 27; Harrison et al., 1992, in Second-line Agents in the Treatment of Rheumatic Diseases, eds. Dixon and Furst, Defcker, New York). Thus, IL-15 plays a significant role in T-cell recruitment and activation in inflammatory polyarthropathy.

295. Triplex forming oligonucleotides (TFO) have been used to inhibit transcription of EL-15 (see U.S. Patent 5,874,566, herein incorporated by reference in its entirety for its teaching of IL- 15 TFOs). Thus, provided is a method for the inhibition of IL- 15 gene transcription, which comprises combining an aminoglycoside dimer, as provided herein, with an oligonucleotide capable of forming a triple-stranded chain in a homopurine/ homopyrimidine region of the IL- 15 gene. Since the transcription of the IL-15 gene is inhibited, this method is effective in inhibiting production of the IL- 15 protein, thus exerting cancer cytotoxic activity.

296. The provided oligonucleotide can correspond to the homopurine/ homopyrimidine region -162/-141 upstream of the IL-15 gene translation initiation point. The provided oligonucleotide can comprise the nucleic acid sequence SEQ ID NO: 80 or 81.

(13) P120

297. Malignant transformation factor pi 20 does not exist in normal tissues in most cases but is present in various cancer tissues. (Cancer Res., 48, 1244-1251 (1988)). Thus, the growth of cancer cells can be inhibited selectively through specific inhibition of the expression of pl20. As a homopurine/homopyrimidine region is present in the transcriptional control region of the gene DNA -1353/-1337 upstream of the translation initiation point, an antigene method in which a triple-stranded chain is formed by winding an antisense molecule or a sense molecule round this homopurine/homopyrimidine region can inhibit expression of the gene (see U.S. Patent 5,869,246, herein incorporated by reference for the teaching of pl20 TFOs).

298. Thus, provided is a method for the inhibition of pl20 gene transcription, which comprises combining an aminoglycoside dimer, as provided herein, with an oligonucleotide capable of forming a triple-stranded chain in a homopurine/ homopyrimidine region of the pi 20 gene. Since the transcription of the pi 20 gene is inhibited, this method is effective in inhibiting production of the pi 20 protein, thus exerting cancer cytotoxic activity.

299. The provided oligonucleotide can correspond to the aforementioned homopurine/homopyrimidine region -1353/-1337 upstream of the pl20 gene translation initiation point, and the -1340 position of the homopurine region is thymine which may be as such or replaced by adenine so that purine bases are continued. The provided oligonucleotide can comprise the nucleic acid sequence SEQ ID NO:82 or 83.

15. Nucleic acid characteristics a) Sequence similarities

300. It is understood that as discussed herein the use of the terms homology and identity mean the same thing as similarity. Thus, for example, if the use of the word homology is used between two non-natural sequences it is understood that this is not necessarily indicating an evolutionary relationship between these two sequences, but rather is looking at the similarity or relatedness between their nucleic acid sequences. Many of the methods for determining homology between two evolutionarily related molecules are routinely applied to any two or more nucleic acids or proteins for the purpose of measuring sequence similarity regardless of whether they are evolutionarily related or not.

301. In general, it is understood that one way to define any known variants and derivatives or those that might arise, of the disclosed genes and proteins herein, is through defining the variants and derivatives in terms of homology to specific known sequences. This identity of particular sequences disclosed herein is also discussed elsewhere herein. In general, variants of genes and proteins herein disclosed typically have at least, about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent homology to the stated sequence or the native sequence. Those of skill in the art readily understand how to determine the homology of two proteins or nucleic acids, such as genes. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level.

302. Another way of calculating homology can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection. I

303. The same types of homology can be obtained for nucleic acids by for example the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. ScL USA 86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306, 1989 which are herein incorporated by reference for at least material related to nucleic acid alignment. It is understood that any of the methods typically can be used and tha;t in certain instances the results of these

various methods may differ, but the skilled artisan understands if identity is found with at least one of these methods, the sequences would be said to have the stated identity, and be disclosed herein.

304. For example, as used herein, a sequence recited as having a particular percent homology to another sequence refers to sequences that have the recited homology as calculated by any one or more of the calculation methods described above. For example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using the Zuker calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by any of the other calculation methods. As another example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using both the Zuker calculation method and the Pearson and Lipman calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by the Smith and Waterman calculation method, the Needleman and Wunsch calculation method, the Jaeger calculation methods, or any of the other calculation methods. As yet another example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using each of calculation methods (although, in practice, the different calculation methods will often result in different calculated homology percentages). b) Hybridization/selective hybridization

305. The term hybridization typically means a sequence driven interaction between at least two nucleic acid molecules, such as a primer or a probe and a gene. Sequence driven interaction means an interaction that occurs between two nucleotides or nucleotide analogs or nucleotide derivatives in a nucleotide specific manner. For example, G interacting with C or A interacting with T are sequence driven interactions. Typically sequence driven interactions occur on the Watson-Crick face or Hoogsteen face of the nucleotide. The hybridization of two nucleic acids is affected by a number of conditions and parameters known to those of skill in the art. For example, the salt concentrations, pH, and temperature of the reaction all affect whether two nucleic acid molecules will hybridize.

306. Parameters for selective hybridization between two nucleic acid molecules are well known to those of skill in the art. For example, in some embodiments selective hybridization conditions can be defined as stringent hybridization conditions. For example, stringency of hybridization is controlled by both temperature and salt concentration of either or

both of the hybridization and washing steps. For example, the conditions of hybridization to achieve selective hybridization may involve hybridization in high ionic strength solution (6X SSC or 6X SSPE) at a temperature that is about 12-25°C below the Tm (the melting temperature at which half of the molecules dissociate from their hybridization partners) followed by washing at a combination of temperature and salt concentration chosen so that the washing temperature is about 5°C to 20°C below the Tm. The temperature and salt conditions are readily determined empirically in preliminary experiments in which samples of reference DNA immobilized on filters are hybridized to a labeled nucleic acid of interest and then washed under conditions of different stringencies. Hybridization temperatures are typically higher for DNA-RNA and RNA- RNA hybridizations. The conditions can be used as described above to achieve stringency, or as is known in the art. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989; Kunkel et al. Methods Enzymol. 1987: 154:367, 1987 which is herein incorporated by reference for material at least related to hybridization of nucleic acids). A preferable stringent hybridization condition for a . DNA:DNA hybridization can be at about 68°C (in aqueous solution) in 6X SSC or 6X SSPE followed by washing at 68°C. Stringency of hybridization and washing, if desired, can be reduced accordingly as. the degree of complementarity desired is decreased, and further, depending upon the G-C or A-T richness of any area wherein variability is searched for. Likewise, stringency of hybridization and washing, if desired, can be increased accordingly as homology desired is increased, and further, depending upon the G-C or A-T richness of any area wherein high homology is desired, all as known in the art.

307. Another way to define selective hybridization is by looking at the amount (percentage) of one of the nucleic acids bound to the other nucleic acid. For example, in some embodiments selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the limiting nucleic acid is bound to the non-limiting nucleic acid. Typically, the non-limiting primer is in for example, 10 or 100 or 1000 fold excess. This type of assay can be performed at under conditions where both the limiting and non-limiting primer are for example, 10 fold or 100 fold or 1000 fold below their k _d , or where only one of the nucleic acid molecules is 10 fold or 100 fold or 1000 fold or where one or both nucleic acid molecules are above their k _d .

308. Another way to define selective hybridization is by looking at the percentage of primer that gets enzymatically manipulated under conditions where hybridization is required to

promote the desired enzyrnatic manipulation. For example, in some embodiments selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75. 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer is enzymatically manipulated under conditions which promote the enzymatic manipulation, for example if the enzymatic manipulation is DNA extension, then selective hybridization conditions would be when at least about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer molecules are extended. Preferred conditions also include those suggested by the manufacturer or indicated in the art as being appropriate for the enzyme performing the manipulation.

309. Just as with homology, it is understood that there are a variety of methods herein disclosed for determining the level of hybridization between two nucleic acid molecules. It is understood that these methods and conditions may provide different percentages of hybridization between two nucleic acid molecules, but unless otherwise indicated meeting the parameters of any of the methods would be sufficient. For example if 80% hybridization was required and as long as hybridization occurs within the required parameters in any one of these methods it is considered disclosed herein.

310. It is understood that those of skill in the art understand that if a composition or method meets any one of these criteria for determining hybridization either collectively or singly it is a composition or method that is disclosed herein. c) Nucleic acids

311. There are a variety of molecules disclosed herein that are nucleic acid based. The disclosed nucleic acids are made up of for example, nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting examples of these and other molecules are discussed herein.

It is understood that for example, when a vector is expressed in a cell, that the expressed mRNA will typically be made up of A, C, G, and U. Likewise, it is understood that if, for example, an antisense molecule is introduced into a cell or cell environment through for example exogenous delivery, it is advantagous that the antisense molecule be made up of nucleotide analogs that reduce the degradation of the antisense molecule in the cellular environment.

312. A nucleotide is a molecule that contains a base moiety, a sugar moiety and a phosphate moiety. Nucleotides can be linked together through their phosphate moieties and sugar moieties creating an internucleoside linkage. The base moiety of a nucleotide can be adenin-9-yl (A), cytosin-1-yl (C), guanin-9-yl (G), uracil- 1-yl (U), and thymin-1-yl (T). The

sugar moiety of a nucleotide is a ribose or a deoxyribose. The phosphate moiety of a nucleotide is pentavalent phosphate. An non-limiting example of a nucleotide would be 3'-AMP (3'- adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate).

313. A nucleotide analog is a nucleotide which contains some type of modification to either the base, sugar, or phosphate moieties. Modifications to the base moiety would include natural and synthetic modifications of A, C, G, and T/U as well as different purine or pyrimidine bases, such as uracil-5-yl (.psi.), hypoxanthin-9-yl (I), and 2-ammoadenin-9-yl. A modified base includes but is not limited to 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenme, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Additional base modifications can be found for example in U.S. Pat. No. 3,687,808, Englisch et al, Angewandte Chemie, International Edition, 1991, 30, 613, and Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain nucleotide analogs, such as 5-substituted pyrimidines, 6-azapyrimidines andN-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine can increase the stability of duplex formation. Often time base modifications can be combined with for example a sugar modifcation, such as 2'-O-methoxyethyl, to achieve unique properties such as increased duplex stability. There are numerous United States patents such as 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941, which detail and describe a range of base modifications. Each of these patents is herein incorporated by reference.

314. Nucleotide analogs can also include modifications of the sugar moiety. Modifications to the sugar moiety would include natural modifications of the ribose and deoxy ribose as well as synthetic modifications. Sugar modifications include but are not limited to the following modifications at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or

unsubstituted ^' Ci to Ci ₀ J ^" alky! or C ₂ to Ci ₀ alkenyl and alkynyl. 2' sugar modiifcations also include but are not limited to -O[(CH ₂ ) _π O] _m CH ₃ , -0(CH ₂ ) _n OCH ₃ , -O(CH ₂ ) _n NH ₂ , -O(CH ₂ ) _n CH ₃ , -0(CH ₂ ) _n -ONH ₂ , and -O(CH ₂ ) _n ON[(CH ₂ ) _n CH ₃ )J ₂ , where n and m are from 1 to about 10.

315. Other modifications at the 2' position include but are not limted to: Ci to C ₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH ₃ , OCN, Cl, Br, CN, CF ₃ , OCF ₃ , SOCH ₃ , SO ₂ CH ₃ , ONO ₂ , NO ₂ , N ₃ , NH ₂ , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Similar modifications may also be made at other positions on the sugar, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Modified sugars would also include those that contain modifications at the bridging ring oxygen, such as CH ₂ and S. Nucleotide sugar analogs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. There are numerous United States patents that teach the preparation of such modified sugar structures such as 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,59-1,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, each of which is herein incorporated by reference in its entirety.

316. Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include but are not limited to those that can be modified so that the linkage between two nucleotides contains a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphonates including 3'-alkylene phosphonate and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. It is understood that these phosphate or modified phosphate linkage between two nucleotides can be through a 3'-5' linkage or a 2'-5' linkage, and the linkage can contain inverted polarity such as 3'-5' to 5V3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included. Numerous United States patents teach how to make and use nucleotides containing modified phosphates and include but are not limited to, 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;

5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is herein incorporated by reference.

317. It is understood that nucleotide analogs need only contain a single modification, but may also contain multiple modifications within one of the moieties or between different moieties.

318. Nucleotide substitutes are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide substitutes are molecules that will recognize nucleic acids in a Watson-Crick or Hoogsteen manner, but which are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to conform to a double helix type structure when interacting with the appropriate target nucleic acid.

319. Nucleotide substitutes are nucleotides or nucleotide analogs that have had the phosphate moiety and/or sugar moieties replaced. Nucleotide substitutes do not contain a standard phosphorus atom. Substitutes for the phosphate can be for example, short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones;formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH ₂ component parts. Numerous United States patents disclose how to make and use these types of phosphate replacements and include but are not limited to 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference.

320. It is also understood in a nucleotide substitute that both the sugar and the phosphate moieties of the nucleotide can be replaced, by for example an amide type linkage (aminoethylglycine) (PNA). United States patents 5,539,082; 5,714,33 l;and 5,719,262 teach how to make and use PNA molecules, each of which is herein incorporated by reference. (See also Nielsen et al., Science, 1991, 254, 1497-1500).

321. It is also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs to enhance for example, cellular uptake. Conjugates can be chemically linked to the nucleotide or nucleotide analogs. Such conjugates include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N. Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium l^-di-O-hexadecyl-rac-glycero-S-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), apolyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), apalmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937. Numerous United States patents teach the preparation of such conjugates and include, but are not limited to U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporated by reference.

322. A Watson-Crick interaction is at least one interaction with the Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute. The Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute includes the C2, Nl, and C6 positions of a purine based nucleotide, nucleotide analog, or nucleotide substitute and the C2, N3, C4 positions of a pyrimidine based nucleotide, nucleotide analog, or nucleotide substitute.

323. A Hoogsteen interaction is the interaction that takes place on the Hoogsteen face of a nucleotide or nucleotide analog, which is exposed in the major groove of duplex DNA. The Hoogsteen face includes the N7 position and reactive groups (NH2 or O) at the C6 position of purine nucleotides.

(1) Sequences

324. There are a variety of sequences related to disclosed herein, these sequences and others are herein incorporated by reference in their entireties as well as for individual subsequences contained therein.

325. It is understood that the description related to this sequence is applicable to any sequence related unless specifically indicated otherwise. Those of skill in the art understand how to resolve sequence discrepancies and differences and to adjust the compositions and methods relating to a particular sequence to other related sequences. Primers and/or probes can be designed for any sequence given the information disclosed herein and known in the art.

(2) Primers and probes

326. Disclosed are compositions including primers and probes, which are capable of interacting with molecules as disclosed herein, hi certain embodiments the primers are used to support DNA amplification reactions. Typically the primers will be capable of being extended in a sequence specific manner. Extension of a primer in a sequence specific manner includes any methods wherein the sequence and/or composition of the nucleic acid molecule to which the primer is hybridized or otherwise associated directs or influences the composition or sequence of the product produced by the extension of the primer. Extension of the primer in a sequence specific manner therefore includes, but is not limited to, PCR, DNA sequencing, DNA extension, DNA polymerization, RNA transcription, or reverse transcription. Techniques and conditions that amplify the primer in a sequence specific manner are preferred. In certain embodiments the primers are used for the DNA amplification reactions, such as PCR or direct sequencing. It is understood that in certain embodiments the primers can also be extended using non-enzymatic techniques, where for example, the nucleotides or oligonucleotides used to extend the primer are modified such that they will chemically react to extend the primer in a sequence specific manner. d) Nucleic Acid Delivery

327. In the methods described above which include the administration and uptake of exogenous DNA into the cells of a subject (i.e., gene transduction or transfection), the disclosed nucleic acids can be in the form of naked DNA or RNA, or the nucleic acids can be in a vector

for delivering the nucleic acids to the cells, whereby the antibody-encoding DNA fragment is under the transcriptional regulation of a promoter, as would be well understood by one of ordinary skill in the art. The vector can be a commercially available preparation, such as an adenovirus vector (Quantum Biotechnologies, Inc. (Laval, Quebec, Canada). Delivery of the nucleic acid or vector to cells can be via a variety of mechanisms. As one example, delivery can be via a liposome, using commercially available liposome preparations such as LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, MD), SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, WI), as well as other liposomes developed according to procedures standard in the art. In addition, the disclosed nucleic acid or vector can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, CA) as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, AZ).

328. As one example, vector delivery can be via a viral system, such as a retroviral vector system, lentivirus, adenovirus, and adeno-associated virus which can package, for example, a recombinant retroviral genome (see e.g., Pastan et al., Proc. Natl. Acad. ScL U.S.A. 85:4486, 1988; Miller et al., MoI. Cell. Biol. 6:2895, 1986). The recombinant retrovirus can then be used to infect and thereby deliver to the infected cells nucleic acid encoding a broadly neutralizing antibody (or active fragment thereof). The exact method of introducing the altered nucleic acid into mammalian cells is, of course, not limited to the use of retroviral vectors. Other techniques are widely available for this procedure including the use of adenoviral vectors (Mitani et al., Hum. Gene Ther. 5:941-948, 1994), adeno-associated viral (AAV) vectors (Goodman et al., Blood 84:1492-1500, 1994), lentiviral vectors (Naidini et al., Science 272:263- 267, 1996), pseudotyped retroviral vectors (Agrawal et al., Exper. Hematol. 24:738-747, 1996). Physical transduction techniques can also be used, such as liposome delivery and receptor- mediated and other endocytosis mechanisms (see, for example, Schwartzenberger et al., Blood 87:472-478, 1996). This disclosed compositions and methods can be used in conjunction with any of these or other commonly used gene transfer methods.

329. As one example, if the antibody-encoding nucleic acid is delivered to the cells of a subject in an adenovirus vector, the dosage for administration of adenovirus to humans can range from about 10 ⁷ to 10 ⁹ plaque forming units (pfu) per injection but can be as high as 10 ¹² pfu per injection (Crystal, Hum. Gene Ther. 8:985-1001, 1997; Alvarez and Curiel, Hum. Gene Titer. 8:597-613, 1997). A subject can receive a single injection, or, if additional injections are necessary, they can be repeated at six month intervals (or other appropriate time intervals, as

determined by the skilled practitioner) for an indefinite period and/or until the efficacy of the treatment has been established.

330. Parenteral administration of the nucleic acid or vector, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein. For additional discussion of suitable formulations and various routes of administration of therapeutic compounds, see, e.g., Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. e) Expression systems

331. The nucleic acids that are delivered to cells typically contain expression controlling systems. For example, the inserted genes in viral and retroviral systems usually contain promoters, and/or enhancers to help control the expression of the desired gene product. A promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site. A promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements.

(1) Viral Promoters and Enhancers

332. Preferred promoters controlling transcription from vectors in mammalian host cells maybe obtained from various sources, for example, the genomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. beta actin promoter. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication (Fiers et al., Nature, 273: 113 (1978)). The immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindm E restriction fragment (Greenway, PJ. et al., Gene 18: 355-360 (1982)). Of course, promoters from the host cell or related species also are useful herein.

333. Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5 ¹ (Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3' (Lusky, MX., et al., Mol. Cell Bio. 3: 1108 (1983)) to the transcription unit. Furthermore, enhancers can be within an intron (Banerji, JX. et al., Cell 33:

729 (1983)) as well as within the coding sequence itself (Osborne, T.F., et al., MoI. Cell Bio. 4: 1293 (1984)). They are usually between 10 and 300 bp in length, and they function in cis. Enhancers function to increase transcription from nearby promoters. Enhancers also often contain response elements that mediate the regulation of transcription. Promoters can also contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression of a gene. While many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, -fetoprotein and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

334. The promotor and/or enhancer may be specifically activated either by light or specific chemical events which trigger their function. Systems can be regulated by reagents such as tetracycline and dexamethasone. There are also ways to enhance viral vector gene expression by exposure to irradiation, such as gamma irradiation, or alkylating chemotherapy drugs.

335. In certain embodiments the promoter and/or enhancer region can act as a constitutive promoter and/or enhancer to maximize expression of the region of the transcription unit to be transcribed. In certain constructs the promoter and/or enhancer region be active in all eukaryotic cell types, even if it is only expressed in a particular type of cell at a particular time. A preferred promoter of this type is the CMV promoter (650 bases). Other preferred promoters are SV40 promoters, cytomegalovirus (full length promoter), and retroviral vector LTF.

336. It has been shown that all specific regulatory elements can be cloned and used to construct expression vectors that are selectively expressed in specific cell types such as melanoma cells. The glial fibrillary acetic protein (GFAP) promoter has been used to selectively express genes in cells of glial origin.

337. Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells) may also contain sequences necessary for the termination of transcription which may affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding tissue factor protein.

The 3' untranslated regions also include transcription termination sites. It is preferred that the transcription unit also contain a polyadenylation region. One benefit of this region is that it increases the likelihood that the transcribed unit will be processed and transported like mRNA. The identification and use of polyadenylation signals in expression constructs is well established.

It is preferred that homologous polyadenylation signals be used in the transgene constructs. In certain transcription units, the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of about 400 bases. It is also preferred that the transcribed units contain other standard sequences alone or in combination with the above sequences improve expression from, or stability of, the construct.

(2) Markers

338. The viral vectors can include nucleic acid sequence encoding a marker product. This marker product is used to determine if the gene has been delivered to the cell and once delivered is being expressed. Preferred marker genes are the E. CoIi lacZ gene, which encodes β-galactosidase, and green fluorescent protein.

339. In some embodiments the marker may be a selectable marker. Examples of suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog. G418, hydromycin, and puromycin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. There are two widely used distinct categories of selective regimes. The first category is based on a cell's metabolism and the use of a mutant cell line which lacks the ability to grow independent of a supplemented media. Two examples are: CHO DHFR- cells and mouse LTK- cells. These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain genes necessary for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media. An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective genes, thus altering their growth requirements. Individual cells which were not transformed with the DHFR or TK gene will not be capable of survival in non-supplemented media.

340. The second category is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection. Examples of such dominant selection use the drugs neomycin, (Southern P. and Berg, P., J. Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid, (Mulligan, R.C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B. et al., MoI. Cell. Biol. 5: 410-413 (1985)). The three examples employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or

neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Others include the neomycin analog G418 and puramycin.

16. Pharmaceutical carriers/Delivery of pharmaceutical products

341. As described above, the compositions can also be administered in vivo in a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.

342. The compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, including topical intranasal administration or administration by inhalant. As used herein, "topical intranasal administration" means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector. Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation. The exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

343. Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.

344. The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via

antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); andRoffler, et al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as "stealth" and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214- 6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits,. enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)). a) Pharmaceutically Acceptable Carriers

345. The compositions, including antibodies, can be used therapeutically in combination with a pharmaceutically acceptable carrier.

346. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid

hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers maybe more preferable depending upon, for instance, the route of administration and concentration of composition being administered.

347. Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

348. Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.

349. The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration maybe topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

350. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

351. Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

352. Compositions Tor oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders maybe desirable..

353. Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines. b) Therapeutic Uses

354. Effective dosages and schedules for administering the compositions may be determined empirically, and. making such determinations is within the skill in the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms disorder are effected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. For example, guidance in selecting appropriate doses for antibodies can be found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389. A typical daily dosage of the antibody used alone might range from about 1 μg/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above.

355. Following administration of a disclosed composition for treating, inhibiting, or preventing a condition or disease, the efficacy of the therapeutic composition can be assessed in various ways well known to the skilled practitioner. For instance, one of ordinary skill in the art will understand that a composition disclosed herein is efficacious in treating or inhibiting an a

disease of ^" condition ϊri"a" subject by observing that the composition reduces or prevents one or more symptoms or characteristics of the disease or condition.

17. Computer readable mediums

356. It is understood that the disclosed nucleic acids and proteins can be represented as a sequence consisting of the nucleotides of amino acids. There are a variety of ways to display these sequences, for example the nucleotide guanosine can be represented by G or g. Likewise the amino acid valine can be represented by VaI or V. Those of skill in the art understand how to display and express any nucleic acid or protein sequence in any of the variety of ways that exist, each of which is considered herein disclosed. Specifically contemplated herein is the display of these sequences on computer readable mediums, such as, commercially available floppy disks, tapes, chips, hard drives, compact disks, and video disks, or other computer readable mediums. Also disclosed are the binary code representations of the disclosed sequences. Those of skill in the art understand what computer readable mediums. Thus, computer readable mediums on which the nucleic acids or protein sequences are recorded, stored, or saved.

357. Disclosed are computer readable mediums comprising the sequences and information regarding the sequences set forth herein.

18. Kits

358. Disclosed herein are kits that are drawn to reagents that can be used in practicing the methods disclosed herein. The kits can include any reagent or combination of reagent discussed herein or that would be understood to be required or beneficial in the practice of the disclosed methods. For example, the kits could include primers to perform the amplification reactions discussed in certain embodiments of the methods, as well as the buffers and enzymes required to use the primers as intended.

19. Compositions with similar functions

359. It is understood that the compositions disclosed herein have certain functions. Disclosed herein are certain structural requirements for performing the disclosed functions, and it is understood that there are a variety of structures which can perform the same function which are related to the disclosed structures, and that these structures will ultimately achieve the same result.

C. Methods of making the compositions

360. The compositions disclosed herein and the compositions necessary to perform the disclosed methods can be made using any method known to those of skill in the art for that particular reagent or compound unless otherwise specifically noted.

1. Nucleic acid synthesis

361. For example, the nucleic acids, such as, the oligonucleotides to be used as primers can be made using standard chemical synthesis methods or can be produced using enzymatic methods or any other known method. Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation (see for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989) Chapters 5, 6) to purely synthetic methods, for example, by the cyanoethyl phosphoramidite method using a Milligen or Beckman System lPlus DNA synthesizer (for example, Model 8700 automated synthesizer of Milligen-Biosearch, Burlington, MA or ABI Model 380B). Synthetic methods useful for making oligonucleotides are also described by Bαita et al., Ann. Rev. Biochem. 53:323-356 (1984), (phosphotriester and phosphite- triester methods)*, and Narang et al., Methods Enzymol., 65:610-620 (1980), (phosphotriester method). Protein nucleic acid molecules can be made using known methods such as those described by Nielsen et al., Bioconjug. Chem. 5:3-7 (1994).

2. Peptide synthesis

362. One method of producing the disclosed proteins is to link two or more peptides, or polypeptides together by protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylrnethyloxycarbonyl) or Boc (tert -butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., Foster City, CA). One skilled in the art can readily appreciate that a peptide or polypeptide corresponding to the disclosed proteins, for example, can be synthesized by standard chemical reactions. For example, a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of a peptide or protein can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group which is functionally blocked on the other fragment. By peptide condensation reactions, these two fragments can be co valently joined via a peptide bond at their carboxyl and amino termini, respectively, to form an antibody, or fragment thereof. (Grant GA (1992) Synthetic Peptides: A User Guide. W.H. Freeman and Co., N.Y. (1992); Bodansky M and Trost B., Ed. (1993) Principles of Peptide Synthesis. Springer-Verlag Inc., NY (which is herein incorporated by reference at least for material related to peptide synthesis). Alternatively, the peptide or polypeptide is independently synthesized in vivo as described herein. Once isolated, these independent peptides or polypeptides may be linked to form a peptide or fragment thereof via similar peptide condensation reactions.

363. For example, enzymatic ligation of cloned or synthetic peptide segments allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains (Abrahmsen L et al., Biochemistry, 30:4151 (1991)). Alternatively, native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two step chemical reaction (Dawson et al. Synthesis of Proteins by Native Chemical Ligation. Science, 266:776-779 (1994)). The first step is the chemoselective reaction of an unprotected synthetic peptide—thioester with another unprotected peptide segment containing an amino-terminal Cys residue to give a thioester-linked intermediate as the initial covalent product. Without a change in the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site (Baggiolini M et al. (1992) FEBS Lett. 307:97-101; Clark-Lewis I et al., J.Biol.Chem., 269:16075 (1994); Clark-Lewis I et al., Biochemistry, 30:3128 (1991); Rajarathnam K et al., Biochemistry 33:6623-30 (1994)).

364. Alternatively, unprotected peptide segments are chemically linked where the bond formed between the peptide segments as a result of the chemical ligation is an unnatural (non-peptide) bond (Schnolzer, M et al. Science, 256:221 (1992)). This technique has been used to synthesize analogs of protein domains as well as large amounts of relatively pure proteins with full biological activity (deLisle Milton RC et al., Techniques in Protein Chemistry IV. Academic Press, New York, pp. 257-267 (1992)).

3. Process claims for making the compositions

365. Disclosed are processes for making the compositions as well as making, the intermediates leading to the compositions. There are a variety of methods that can be used for making these compositions, such as synthetic chemical methods and standard molecular biology methods. It is understood that the methods of making these and the other disclosed compositions are specifically disclosed.

366. Disclosed are cells produced by the process of transforming the cell with any of the disclosed nucleic acids. Disclosed are cells produced by the process of transforming the cell with any of the non-naturally occurring disclosed nucleic acids.

367. Disclosed are any of the disclosed peptides produced by the process of expressing any of the disclosed nucleic acids. Disclosed are any of the non-naturally occurring disclosed peptides produced by the process of expressing any of the disclosed nucleic acids. Disclosed are any of the disclosed peptides produced by the process of expressing any of the non-naturally disclosed nucleic acids.

368. Disclosed are animals produced by the process of transfecting a cell within the animal with any of the nucleic acid molecules disclosed herein. Disclosed are animals produced by the process of transfecting a cell within the animal any of the nucleic acid molecules disclosed herein, wherein the animal is a mammal. Also disclosed are animals produced by the process of transfecting a cell within the animal any of the nucleic acid molecules disclosed herein, wherein the mammal is mouse, rat, rabbit, cow, sheep, pig, or primate.

369. Also disclose are animals produced by the process of adding to the animal any of the cells disclosed herein.

D. Methods of using the compositions

1. Methods of using the compositions as research tools

370. The disclosed compositions can be used in a variety of ways as research tools. For example, the disclosed compositions, can be used to study the interactions between aminoglycosides and nucleic acids, by for example acting as inhibitors of triplex binding.

371. The compositions can be used for example as targets in combinatorial chemistry protocols or other screening protocols to isolate molecules that possess desired functional properties related to their nucleic acid binding.

372. The disclosed compositions can be used as discussed herein as, either reagents in micro arrays or as reagents to probe or analyze existing microarrays. The disclosed compositions can be used in any known method for isolating or identifying single nucleotide polymorphisms. The compositions can also be used in any method for determining allelic analysis. The compositions can also be used in any known method of screening assays, related to chip/micro arrays. The compositions can also be used in any known way of using the computer readable embodiments of the disclosed compositions, for example, to study relatedness or to perform molecular modeling analysis related to the disclosed compositions.

E. Examples

373. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.

1. Example 1. From triplex to B-form duplex stabilization: reversal of target selectivity by aminoglycoside dimers

374. The poly(dA):poly(dT) triplex melt is seen at 34 degrees C and the duplex melts at 71 degrees C (15OmM KCl, pH6.8 Fig. 28) (Arya, D. P.;Co.ee, R. L., Jr.;Willis, B.; Abramovitch, A. I. J. Am. Chem. Soc. 2001, 123, 5385-5395). Upon increasing neomycin concentrations, the triplex melt increases without any effect on the duplex melt. (Arya, D. P.;Co.ee, R. L., Jr.;Willis, B.; Abramovitch, A. I. J. Am. Chem. Soc. 2001, 123, 5385-5395, Arya, D. P.;Co.ee, R. L., Jr. Bioorg. Med. Chem. Lett. 2000, 10, 1897-1899). When a small amount of the neomycin— neomycin dimer was added to this triplex, the UV melts showed a remarkably surprising pattern (Fig. 28). The hypochromicity observed for the triplex melt simply decreases with a concomitant disappearance of the transition at a slightly higher dimer concentration (rdb=0.13, where rdb is the ratio of the drug to the DNA). On the other hand, the duplex melt increases by 8 degrees C. To confirm that this behavior was not limited to the poly(dA):poly(dT) structure, the smaller duplex dT(16):dA(16) was looked at. A Job plot of dA16 and dT16 shows the difference in selectivity for neomycin versus neomycin-tobramycin dimer at low temperature (Fig. 29). While the minimum is clearly seen at 66% dT16 in the presence of neomycin, neomycin-tobramycin dimer shows no such preference, but leads to a stabilization of the duplex such that a clear minimum between duplex/triplex is not distinguishable. Both dimers show similar UV thermal melt patterns at low concentrations.

375. Isothermal titration calorimetry was then carried out with neomycin-tobramycin dimer and poly(dA):poly(dT) duplex (Fig. 29 a,b; Table 3) as well as the poly-(dA):2ρoly(dT) triplex. A complete reversal of binding modes as seen for neomycin is seen with this dimer. Neomycin shows nonspecific electrostatic binding with the DNA duplex and a single high affinity binding site with the DNA triplex; neomycin— tobramycin dimer however, shows a high affinity binding site with the DNA duplex, mainly driven by a large negative enthalpy. While neomycin gives a high association constant with the poly-(dA):2(dT) triplex (4.5 base triplets/drug binding site) (Arya, D. P.;Micovic, L.;Charles, I.;Co.ee, R. L., Jr.;Willis, B.;Xue, L. J. Am. Chem. Soc. 2003, 125, 3733-3744), neomycin-tobramycin dimer leads to a K _a of 1.0-10 ⁸ M- ¹ in binding to the poly(άA):(dT) duplex (7 base pair/drug). Multiple binding sites were observed in titration of the dimer to the poly(dA):2poly(dT) triplex, which could not be fit to available models. Additionally, titration of neomycin-neomycin dimer (Fig. 30 c,d, Table 2) to the poly(dA):(dT) duplex yields an association constant similar to that observed with the neomycin-tobramycin dimer (with an even higher enthalpy contribution to binding). This

suggests that the shape/charge complementarity to the duplex groove is perhaps more important than specific atom contacts made by any ligand.

376. Table 2. ITC-Derived Thermodynamic Profiles for the Binding of neomycin conjugates to poly(dA).poly(dT) double helix in 10 mM sodium cacodylate, 0.5 mM EDTA, 150 mM KCl, pH 6.8 at 20°C.

377. What then is the cause of this surprising duplex stabilization by these dimers: The major groove of the duplex remains a plausible binding site, and is substantiated by the following observations: (1) triplex destabilization at low drug concentrations (blocking the third strand from the major groove. (2) No stabilization of the DNA duplex by neomycin or tobramycin (indicating that these ligands do not occupy the DNA minor groove) (Arya, D. P.;Co.ee, R. L., Jr.;Willis, B.; Abrarnovitch, A. I. J. Am. Chem. Soc. 2001, 123, 5385-5395). (3) A larger binding site (7-10 base pairs) and a good charge/shape complementarity of the dimer to the DNA major groove. The dimeric conjugate can take a conformation mimicking the triplex third strand such that the two ends of the groove are held together by H-bonds/electrastatic complementarity (neomycin alone is unable to do that and has been shown to have a better charge/shape complementarity to the triplex W-H groove or other A-form major grooves). (Arya, D. P.;Micovic, L.;Charles, I.;Co.ee, R. L., Jr.; Willis, B.;Xue, L. J. Am. Chem. Soc. 2003, 125, 3733-3744). Few carbohydrate ligands have ever been known to show such high-affinity binding to duplex structures. (Nicolaou, K. C.;Smith, B. M.;Ajito, K.;Komatsu, H.; Gomez- Paloma, L.;Tor, Y. J. Am. Chem. Soc. 1996, 118, 2303-2304). Major groove DNA binding ligands (unlike minor groove binders) are limited to protein structures and a few small ligands. Neomycin (and other aminoglycosides) can stabilize DNA/RNA triplexes, hybrid duplexes, hybrid triplex and even stabilize tetraplexes (Arya, D. P.;Co.ee, R. L., Jr.;Willis, B.; Abramovitch, A. I. J. Am. Chem. Soc. 2001, 123, 5385-5395; Arya, D. P.;Co.ee, R. L., Jr.;Charles, I. J. Am. Chem. Soc. 2001, 123, 11093-11094; Arya, D. P.;Xue, L.; Tennant, P. J. Am. Chem. Soc. 2003, 125, 8070-8071; Arya, D. P.;Micovic, L.;Charles, I.;Co.ee, R. L., Jr.; Willis, B.;Xue, L. J. Am. Chem. Soc. 2003, 125, 3733-3744; Arya, D. P.;Xue, L.;Willis, B. J. Am. Chem. Soc. 2003, 125, 10148-10149). While it stabilizes DNA triplex structures, neomycin does not affect DNA duplex stability (under physiological ionic conditions). (Arya, D. P.;Co.ee,

R. L., Jr.;Willis, B.; Abramovitch, A. I. J. Am. Chem. Soc. 2001, 123, 5385-5395). It has also suggested that aminoglycoside specificity (neomycin in high nM-lowlM range) may be for nucleic acid forms that show some features characteristic of an A-type conformation (RNA triplex, DNA-RNA hybrid duplex, RNA duplex, DNA triplex, A-form DNA duplex, and DNA tetraplex), rather than for naturally occurring RNA. (Arya, D. P.;Xue, L.;Willis, B. J. Am. Chem. Soc. 2003, 125, 10148-10149). Neomycin fits better in the narrower A-form major groove but does not have a good charge or shape complementarity to the major groove of B-form DNA. It has been shown that a Hoechst— neomycin conjugate can force neomycin into the larger B-form DNA duplex groove. (Arya, D. P.;Willis, B. J. Am. Chem. Soc. 2003, 125, 12398-12399) In retrospect, the larger size of the B-form major groove could be a surprisingly good fit for dimeric aminoglycosides. (Michael, K.;Wang, H.;Tor, Y. Bioorg. Med. Chem. 1999, 7, 1361-1371). Groove recognition of A and B-form duplexes and triplexes, and even higher order structures can then be made possible if one carefully applies the principles of charge/shape complementarity to nucleic acid recognition.

378. Aminoglycosides, with their unique positively charged manifold disclosed herein present just such a new motif of recognition for these higher order RNA/DNA nucleic acid forms. The results of the disclosed experiments described herein identify a new duplex groove binding ligand selective for B-form DNA duplexes.

2. Example 2. Design of sequence specific Neo-Neo-Hoechst Conjugate

379. The structural features of the different major grooves dictate the recognition by small molecules. Aminoglycosides bind within the deep and narrow RNA major groove due to favorable shape and charge complementarity. The B-DNA major groove has been a difficult target for small molecules due to a significantly wider and shallower major groove. To date, the majority of carbohydrate interactions with DNA involve interactions within the minor groove. Among the classes of compounds known for DNA binding are enediyne antibiotics, anthracyclines, pluramycins, indolocarbazoles, and aureolic acids. Of the limited number of carbohydrates known and studied for DNA binding, only a select few displayed major groove contacts. The majority of compounds within this small group are known for dual groove (both minor and major) interactions. These interactions are often accompanied or assisted by a central intercalative structure, known to display a variety of binding characteristics, as DNA cleavage agents (neocarzinostatin), alkylating agents (altromycin B), or tandem intercalative-groove binding ligands (nogalamycin, respinomycin, NB-506). A target for proteins, the B-DNA groove remains an attractive target. The observation that dimeric aminoglycosides bind B-DNA

duplexes provides a good starting point for developing ligands to target the B-DNA major groove.

380. Disclosed is the multi-recognition of B-DNA by conjugating neomycin with the minor groove binding ligand Hoechst 33258. Hoechst 33258 is a well known minor groove binding ligand with particular affinity for A/T bases. Conjugates of Hoechst with other DNA - binding moieties has indicated enhancements in recognition over Hoechst 33258 alone. These include conjugates with porphyrins (Frau, S., et al. Bioconj. Chem. 1997, 8, 222-23 l)(Frau, S., et al. Nucleos. Nucleotid. Nucleic Acids 2001, 20, 145-156), polyamides (Satz, A. L., et al. Bioorg. Med. Chem. 2002, 10, 241-252)(Satz, A. L., et al. J. Am. Chem. Soc. 2001, 123, 2469-2477), polyamines (Satz, A. L., et al. Bioorg. Med. Chem. Lett. 2000, 8, 1871-1880)(Satz, A. L., et al. Bioorg. Med. Chem. Lett. 1999, 9, 3261-3266), and DNA (Wiederholt, K., et al. Nucleosides Nucleot. 1998, 17, 1895-1904)(Wiederholt, K., et al. J. Am. Chem. Soc. 1996, 118, 7055- 7062)(Rajur, S. B., et al. J. Org. Chem. 1997, 62, 523-529)(Robles, J., et al. J. Am. Chem. Soc. 1997, 119, 6014-6021). Such conjugates have been shown to significantly enhance DNA stability and extend the sequence specific binding site size up to ten and 12 A/T base pairs. Disclosed herein are dimeric aminoglycoside conjugation with Hoechst 33258.

381. Molecular modeling studies indicate both minor groove and major groove interactions by the Hoechst and neomycin dimers, respectively. With significant linker flexibility and length, the interactions between the respective moieties can be virtually unperturbed, hi one design, Methylene glycol derivatives were used to constitute the linkages between binding regions. Along with commercially availability of starting materials and published synthetic routes to crucial intermediates, such linkers conveniently provide the appropriate extension to desirable binding sites.

382. For the synthesis of the novel neo-neo-Hoechst conjugate (termed NNH), first a Mitsunobu-type reaction was carried out of Methylene glycol monotosylate with p-cyanophenol.

Terminal substitution of the tosylate with mono-protected diamine provided the secondary amine, which could be subsequently protected as a trifiuoroacetamide using trifiuoroacetic anhydride in pyridine. With both amines protected, anhydrous HCl in dry methanol provided the methyl imidate ester, which could be coupled with diamine in a solution of acetic acid and dry methanol. The choice of dry methanol in both imidation and coupling steps was due to coupling difficulties with the ethyl (alcohol). It was later discovered that trifluoroacetyl deprotection was most likely occurring in the coupling step. This was indeed confirmed when dry methanol was used. The coupling reaction was successful. The low yield (28%) in the coupling step may

likely stem from competing reactions with deprotected amines. Nonetheless, the synthesis of Hoechst-diamine derivative 5 was successful, as indicated by ¹ H ISIMR and MALDI-MS. The synthetic route for the Hoechst-diamine is outlined in Figure 31.

383. The reaction of diamine 5 with neomycin isothiocyanate 6 (Figure 32) proceeded at elevated temperatures due to the somewhat diminished nucleophilicity of the 2° amine. The resulting N-Boc protected conjugate was then deprotected under standard TFA/CH ₂ C1 ₂ conditions using ethanedithiol as a scavenger due to the significant number of Boc protecting groups in the molecule. The final compound 8 (NNH) was isolated as a trifluoroacetate salt after lyophilization. a) Spectroscopic Studies

384. The thermal stability of poly(dA)»poly(dT) in the presence of NNH was first investigated. It was found that the conjugate displayed a marked effect on the stability of poly(dA)»poly(dT) duplex when compared to both that in the absence of ligand. There is a noticeable shift in the triplex transition as concentrations of NNH are increased. Furthermore, the dissociation of duplex DNA in the presence of NNH occurs at a higher temperature (~85° C), upwards of 15 ⁰ C higher than duplex alone (72 ⁰ C).

385. The UV melting studies were extended to include oligomeric DNA duplex as well, with ClA ₂₂ ^dT ₂₂ duplex. In the absence of ligand, these duplexes denature at 51 oC. However, when NNH is introduced, noticeable shifts in the melting transition are apparent (broad transition melting over a 25 ⁰ C range past 7O ⁰ C), signifying key favorable interactions between the ligand and DNA. b) Organic Synthesis

386. Compound 2. A solution of p-cyanophenol (200 mg, 1.68 mmol), Methylene glycol monotosylate (1.02 g, 3.35 mmol), and triphenylphosphine (870 mg, 3.35 mmol) in dioxane (25 mL) was stirred at room temperature while DIAD (665 μL, 3.35 mmol) was added dropwise over a period of 30 minutes. After overnight stirring, the dioxane was removed before further co-evaporations with toluene (3 x 10 mL) and purification over silica gel column (0 to 100% ether in hexane) to afford 534 mg (78%) product. R _f 0.56 (8:2 EtOAc:hexane); ¹ H NMR (500 MHz, CDCl ₃ ): δ 7.73 (d, 2H, J = 8.3), 7.52 (d, 2H, J = 9.2), 7.28 (d, 2H, J = 8.3), 6.91 (d, 2H, J = 9.2), 4.08-4.10 (m, 4H), 3.79 (t, 2H, J = 4.8), 3.60-3.64 (m, 4H), 3.55-3.57 (m, 2H), 2.37 (s, 3H); ¹³ C (125 MHz, CDCl ₃ ): δ 162, 145, 134, 133, 130, 128, 115, 104, 71, 69.5, 69.3, 68.9, 67.8, 22; MS (MALDI-TOF) m/z for C ₂₀ H ₂₃ NNaO ₆ S [M+Na] ⁺ 428.70, found 428.47.

387. Compound 3. Mono-protected diamine (458 mg, 1.9 mmol) was mixed with tosylate 2 (190 mg, 0.47 mmol), K ₂ CO ₃ (150 mg, lmmol), and KI (16 mg, 0.01 mmol) in DMF containing 3 angstrom molecular sieves and heated at 70 ⁰ C under argon overnight. DMF was evaporated under high vacuum (50 ⁰ C) and the residue was dissolved in CH ₂ Cl ₂ and filtered before concentration and loading onto a column of silica and purification using a gradient of MeOH (0 to 15%) in CH ₂ Cl ₂ to afford 136 mg (61%) of product as an oil. R _f 0.44 (85:15:0.1 CH ₂ Cl ₂ :MeOH:NH ₄ OH); ¹ H NMR (500 MHz, CDCl ₃ ): δ 7.52 (d, 2H, J = 8.7), 6.92 (d, 2H, J = 9.2), 4.11 (m, 2H), 3.80 (m, 2H), 3.40-3.75 (m, 16H), 2.99 (m, 4H); ¹⁹ F (500 MHz, CDCl ₃ ): - 75.5; MS (MALDI-TOF) m/z for C ₂₁ H ₃₀ F ₃ N ₃ O ₆ [M+H] ⁺ 478.47, found 428.70.

388. Compound 4. To a solution of 3 (180 mg, 0.377 mmol) in dry pyridine (2 niL) and triethylamine (1 mL) was added trifluoroacetic anhydride (100 μL, 0.64 mmol) dropwise at O ⁰ C The solution was brought to room temperature and allowed stirring overnight. Solvents were removed by rotary evaporation _* with co-evaporations with toluene (2 x 5 mL) and drying under high vacuum (50 ⁰ C). After addition of diethyl ether, the pyridinium salt was filtered. Further salt was removed by rinsing the organics with cold water (3 5 mL) and brine (1 x 5 mL).

Trace NEt ₃ was removed by purifying the syrup over silica gel using a gradient of ethyl acetate (up to 10%) in ether. Yield was 125 mg (58%). R _f 0.53 (95:5 CH ₂ Cl ₂ :Me0H). ¹ H NMR (500 MHz, CDCl ₃ ) δ 7.57 (d, 2H, J = 8.3), 6.95- (d, 2H, J = 8.7), 4.13 (m, 2H), 3.81 (m, 2H), 3.75-3.50 (m, 20H); ¹⁹ F (500 MHz, CDCl ₃ ): -68.5, -75.7.

389. Compound 5. Anhydrous HCl was rapidly bubbled through a solution of 4 (56 mg, 98 μmol) in dry methanol (3 mL) at 0 ⁰ C for 20 minutes. The solution was then sealed sufficiently and stored at 4 ⁰ C overnight. TLC showed the absence of starting material and a . more polar product. After bringing to room temperature, the gas was removed by purging with nitrogen into an aqueous sodium bicarbonate bath, and ethanol was evaporated by rotary evaporation. After liberal washes with ether, the resulting crystalline solid was dried under high vacuum to yield 63 mg (quant.) of product, which was used immediately in the coupling step without characterization.

390. Freshly prepared 4-[5-(4-Methyl-piperazin-l-yl)-lH-benzimidazol-2-yl]-benzene - 1,2-diamine (10 mg, 31 μmol) and imidate methyl ester of 4 (21 mg, 34 μmol) in dry MeOH (2 mL) glacial acetic acid (0.5 mL) was heated to reflux for 6 hours. TLC indicated products characteristic of Hoechst compounds (bright yellow fluorescence). A faint amount of the trifluoroacetyl - protected product was observed (Rf 0.33 in 1 : 1 CH ₂ Cl ₂ MeOH with trace NEt ₃ ). A more polar product was later identified (after purification) as the de-trifluoroacetylated

product. The reaction solution was concentrated and loaded onto a silica gel column before purification: gradient of MeOH in EtOAc followed by a 10:10:1 mixture of acetone:MeOH:NEt ₃ to push off the free diamine product, which eluted as a bright yellow-green fluorescent product of sufficient purity. Triethylamine in the eluted product was removed by rinses with CH ₂ Cl ₂ . Yield (deprotected product): 6 mg (28%); ¹ H NMR (500 MHz, CD ₃ OD) δ 8.28 (s, IH), 8.09 (d, 2H, J = 8.7), 7.95 (d, IH), 7.71 (d, IH, J),= 8.3), 7.52 (d, IH, J - 8.7), 7.15 (m, 3H), 7.07 (d, IH), 4.24 (m, 2H), 3.80-3.60 (m, 12H), 3.30 (m, 4H), 3.00 (m, 4H), 2.77 (m, 4H), 2.70 (m, 4H), 2.38 (s, 3H); MS (MALDI-TOF) m/z for C ₃₇ H ₅₀ N ₈ NaO ₅ [M] ⁺ 686.84, found 686.96.

391. Compound 7 (Boc-protected Neo-Neo-Hoechst 33258 conjugate). A solution of diamine 5 (3 mg, 4.3 μmol) in dry pyridine (2 mL) containing DMAP (1 mg) was added to a dry flask containing neomycin isothiocyanate 7 (10 mg, 7.6 mmol) at room temperature. The solution was allowed to stir under argon overnight at 45 ⁰ C. TLC indicated the presence of a fluorescent product, also positive for anisaldehyde staining/charring. The pyridine was evaporated, followed by co-evaporation with toluene (2 x 2 mL), and chromatography over silica (gradient of MeOH in CH ₂ Cl ₂ ) afforded 6.5 mg (45%) of a light brown solid. Rf= 0.50 in 86:14 CH ₂ Cl ₂ :Me0H. ¹ H NMR (500 MHz, D ₂ O): 8.2Q (s, IH,), 8.00 (d, 2H), 7.89 (d, IH), 7.64 (b, IH), 7.42 (b, IH), 7.09 (d, 2H), 6.98 (m, 2H), 6.42 (b, IH), 5.27 (m, 2H), 5.00 (m, 2H), 4.17- 3.00 (36H), 2.90-2.70 (8H), 2.59 (m, 4H), 2.29 (s, 3H), 2.00-1.80 (m, 4H), 1.43 (m, 108H). MS (MALDI-TOF) m/z for C ₁₄₉ H ₂₄₄ N ₂₂ O ₅₃ [M] ⁺ 3319.91, found 3319.88.

392. Compound 8 (Neo-Neo-Hoechst 33258 conjugate). A solution of 7 (6.5 mg, 1.96 μmol) in 1 mL of 1:1 CH ₂ C1 ₂ :TFA containing trace ethanedithiol (1 μL) stirred at room temperature for 5 hours. Solvents were evaporated before dissolution in dl H ₂ O (2 x 1 mL), filtering over 0.2 μm filter, freezing and lyophilization to give 6.4 mg (85%); NMR proved difficult due to very low concentrations. MS (MALDI-TOF) m/z [M+H] ⁺ 2119.52, calcd for C ₈₉ H ₁₄₉ N ₂₂ O ₂₉ S ₄ 2119.37. c) Materials and methods (1) Nucleic Acids

393. The concentrations of nucleotide solutions were determined using the extinction coefficients (per mol of nucleotide) calculated according to the nearest neighbor method. The concentrations of all the polymer solutions were determined spectrophotometrically using the following extinction coefficients (in units of mol of nucleotide/L ^"1 cm ^"1 ): ε ₂₆₅ = 9000 for poly(dT), ε ₂₆₀ = 6000 for poly(dA).poly(dT), ε ₂₅₃ = 7400 for poly(dG).poly(dC), ε ₂₇₄ = 7400. All oligonucleotides were synthesized on an Applied Biosystm 8890 using standard

phosphoramidite chemistry. Oligonucleotides were purified by ion-exchange HPLC on a Gen- Pak FAX (4.6 x 100 mm) ion exchange column, eluting with Buffer A (25 mM tris.HCl, 1 rnM EDTA, 10 % CH ₃ CN, pH 8.0) from 98% to 50% and Buffer B (25 mM tris-HCl, 1 mM EDTA, 1 M NaClO ₄ , 10 % CH ₃ CN, pH 8.0) from 2% to 50% in 15 minutes. In all cases where mentioned, the term ra _b refers to molar ratio of drug to base.

(2) UV Spectroscopy

394. All UV spectra were recorded on a Cary 100 Bio UV/Vis spectrophotometer equipped with a thermoelectrically controlled 12-cell holder. Quartz cells with a 1 cm pathlength were used for all the absorbance studies. Spectrophotometer stability and λ alignment were checked prior to initiation of each melting point experiment. In all oligonucleotide experiments, the samples were heated to 95°C for 5 minutes, and then slowly cooled to 4°C and incubated for 16 hrs. The temperature was raised in 0.2°C/min increments. For all polynucleotide experiments, the samples were heated to 95 ⁰ C followed by annealing at a rate of 0.2°C/min, then melting at 0.2°C/min. Samples were brought back to 20°C after run. All UV melting experiments were monitored under 260, 280, and 284 nm. For the T _n , determinations, derivatives were used. Data were recorded every 1.0°C.

(3) Isothermal Titration Calorimetry (ITC)

395. Isothermal calorimetric measurements were performed at 2O ⁰ C on a MicroCal VP-ITC (MicroCal, Inc.; Northampton, MA). In a typical experiment, 5 μL aliquots of 440 μM neomycin-neomycin conjugate were injected from a 250 μL rotating syringe (300 rpm) into an isothermal sample chamber containing 1.42 niL of a polynucleotide duplex solution that was 60 μM/bp. Each experiment of this type was accompanied by the corresponding control experiment in which 5 μL aliquots of 440 μM drug were injected into a solution of buffer alone. The duration of each injection was 10 s, and the delay between injections was 300 s. The initial delay prior to the first injection was 60 s. Each injection generated a heat burst curve (microcalories per second vs seconds). The area under each curve was determined by integration using the Origin (version 5.0) software to obtain a measure of the heat associated with that injection. The heat associated with each drug-buffer injection was subtracted from the corresponding heat associated with each drug-DNA injection to yield the heat of drug binding for that injection. The resulting ITC profile is shown in Figure 34.

3. Example 3 Binding duplex RNA a) Materials and Methods

396. Nucleic acids: RNA polymers (polyA, lot no. 3104110011; polyU, lot no. 4034440021) were purchased from Pharmacia Biotech (Piscataway, NJ). Concentrations were determined by UV absorbance using ε ₂₅₈ = 9800 (M ⁴ Cm ^"1 ) for polyA and ε ₂₆₀ = 9350 (M ^"1 Cm ^'1 ) for polyU. For all experiments with polymeric RNA, solutions of individual strands were dialyzed extensively (48 hrs.) against buffer using SpectraPor Float-A-Lyzer dialysis units, MW cutoff 3500 Da (Spectrum Labs, Rancho Dominguez, CA) before quantitation. The self- complementary oligomers r(CGCAAAUUUGCG) (SEQ ID NO:88) and r(CGCAAGCUUGCG) (SEQ ID NO: 89) were purchased from Dharmacon (Lafayette, CO) and deprotected using standard protocols provided by the supplier. Duplex RNA was formed by heating at 95 ⁰ C for 5 minutes before slowly annealing to room temperature and storage at 4 ⁰ C between experiments. Solutions for all experiments were in buffer consisting of 10 rnM PIPES, 1 mM EDTA, 100 mM NaCl, pH 7.0 unless otherwise noted.

397. Chemicals: Neomycin B (sulfate salt) was purchased from ICN Biomedicals and used without further purification. Piperidinesulfonic acid (PIPES) and NaCl were purchased from Fisher Scientific. Hoechst 33258 was purchased from Acros Organics. Conjugate NHl was synthesized as reported previously (Arya, D. P., et al. J. Am. Chem. Soc. 2003, 125, 12398- 12399). Quantitation of Hoechst 33258 (ε ₃₃₈ - 42,000 M ^-1 Cm ^"1 ) and NHl (ε ₃₄₂ = 39,241 M ^-1 Cm ^{" l} ) in aqueous solutions was done using UV/Vis absorbance. To ensure stability and minimum adsorption of Hoechst compounds to container walls, all solutions were stored in non- transparent, polystyrene tubes.

398. UV Melting: All experiments were carried out using a Cary IOOE UV/Vis spectrophotometer equipped with a thermoelectrically controlled 12-cell holder. All samples were analyzed in quartz cells (1 cm pathlength). Lamp stability and wavelength alignment were checked prior to each experiment. Unless otherwise noted, prior to analysis (heating from 20-95 ⁰ C at a rate of 0.2 deg/min., monitoring at 260 nm), samples were prepared in polystyrene tubes and heated at 95 ⁰ C for 5 minutes before slowly annealing to room temperature. In all experiments, [polyA»polyU] = 20 μM/base pair. Melting temperature (T _m ) assignments were done using first derivative analysis provided by the Cary software.

399. Isothermal Titration Calorimetry (ITC): Measurements were performed at 20 ⁰ C on a MicroCal VP-ITC (MicroCal, Inc., Northampton, MA). In a typical experiment, 10 μL aliquots of ligand (150 μM for neomycin, 100 μM for NHl) were injected from a 250 μL

rotating syringe (300 rpm) into an isothermal sample chamber containing 1.42 mL of polyA.polyU duplex solution that was 40 μM/bp. Each experiment of this type was accompanied by the corresponding control experiment in which 10 μL aliquots of identical drug solutions were injected into a solution of buffer alone. The duration of each injection was 10 s, and the delay between injections was 300 s. The initial delay prior to the first injection was 60 s. Each injection generated a heat burst curve (microcalories per second vs. seconds). The area under each curve was determined by integration using Origin (version 5.0) software to obtain a measure of the heat associated with that injection. The heat associated with each drug-buffer injection was subtracted from the corresponding heat associated with each drug-DNA injection to yield the heat of drug binding for that injection. Binding isotherms were fit using an identical site binding model in the Origin software provided by MicroCal.

400. For NHl experiments, ligand-into-buffer experiments displayed a positive (endothermic) δH accompanied for each injection, with a slight decrease as concentration in the sample chamber increased. This is attributed to the ligand's propensity to aggregate at micromolar concentrations (often seen with Hoechst compounds). Hoechst 33258 self- association is well known (for example, see Biochemistry 1990, 29, 9029-9039). The decrease in δH is most likely due to free ligand self-association (an exothermic interaction) as concentrations increase inside the sample chamber, accompanied with the ligand-ligand dissociation (endothermic) once the concentrated solution is injected. Hoechst 33258 displays a pronounced effect when titrated into aqueous solution, with a drastic change from positive to negative δH as concentrated ligand is serially titrated into aqueous buffer. In the present case, the following assumption was made to fit the observed binding curve: given the high binding of neomycin to RNA, all ligand that is introduced to the host RNA duplex becomes completely bound, such that δH solely represents the ligand-ligand dissociation and subsequent binding to RNA. Therefore, the initial (first) peak representing ligand-into-buffer δH is used for data subtraction from the RNA-ligand titration. This assignment is supported by two events. (1) δH values for excess-site binding (approx. first 7 injections) are nearly identical. Had there been ligand-ligand interaction, δH values should decrease (RNA-ligand and ligand-ligand binding) in this excess-site region of the titration curve, since it is observed in the ligand-into-buffer titration. (2) The data fit a theoretical model with very little error. Such an argument has been introduced before with Hoechst 33258 binding to DNA analyzed via fluorescence (Biochemistry 1990, 29, 9029-9039).

401. Fluorescence titrations: Equilibrium binding experiments were done using a Photon Technology International instrument (Lawrenceville, NJ) at ambient (22 ⁰ C) temperature.

A solution of NHl (serially diluted to working concentrations) was excited at 342 nm (slits = 3 nm) and resulting emission curves (from 390-600 nm) were recorded after serial additions of a concentrated RNA solution (ρoly»polyU was 543 μM, r(CGCAAAUUUGCG) ₂ (SEQ ID NO:88)was 60 μM, and r(CGCAAGCUUGCG) ₂ (SEQ BD NO:89) was 40 μM). After each addition, the solution was mixed by pipetting up and down with a Pasteur pipette treated with silanizing agent (Sigmacote) to avoid ligand adsorption to the glass. Sample equilibrium was monitored by continually exciting/scanning the sample at different times, and was usually reached within 2 minutes. A silanized (SigmaCote) cuvette was used in all experiments. All data were normalized to account for the (small) dilution of sample upon addition of substrate.

402. CD Spectropolarimetry titrations: Circular dichroism (CD) experiments were done at 20 ⁰ C using a Jasco J-810 spectropolarimeter. A concentrated solution of ligand (500 μM neomycin or NHl) was added to a 40 μM solution of polyA«polyU and allowed to stir constantly before scanning from 350-220 nm. As with fluorescence experiments, equilibrium was determined by periodically scanning the sample over a period of time (up to 10 minutes) for the first few additions of ligand, and was reached within 3 minutes. AU data were normalized to account for the (small) dilution of sample upon addition of ligand.

403. Viscometry: Viscosity measurements were conducted using a Cannon-Ubbelohde 75 capillary viscometer submerged in a water bath at 27 + 0.05 ⁰ C. Flow times of buffer only followed by RNA (1030 μL of 100 μM in base pair) were recorded in triplicate before titrations of concentrated ligand solutions (500 μM) with mixing by bubbling of air (using a pipette bulb) through the solution. Flow times after each titration were recorded in triplicate. In all cases, standard deviations were less than 0.1 s. AU solutions were in PIPESlO buffer (10 mM PIPES, 1 mM EDTA, pH 7.0) containing 100 mM NaCl. Flow times for buffer alone were in the range of 106 s, whereas RNA alone was approx. HO s. Flow times for ligand titrations into RNA ranged from 110 to 107 s. The viscosity for each titration was determined using the relationship (Bordelon, J. A., et al. J. Phys. Chem. B 2002, 106, 4838-4843)

404. where L is length of RNA complexed with ligand, L ₀ is length of DNA/RNA alone, η is intrinsic viscosity, t is flow time of complex, t _b is flow time of buffer, and t ₀ is flow

time of RNA alone. Data were plotted in the form of relative viscosity, L/L ₀ ,versus r (ratio of bound ligand to KNA concentrations) with a comparison to theoretical intercalation (1 + r versus r) to convey the differences in ligand binding to that of intercalation.

405. Computer Modeling: The DNA duplex d(CGCAAATTTGCG) ₂ (SEQ ID NO:88) was extracted from pdb entry 296d, RNA duplex r(CGCAAATTTGCG) ₂ (SEQ ID NO:88) was obtained from PDB entry Ial5 (Conte, M. R., et al. Nucleic Acids Res. 1996, 24, 3693-3699), and r(CGCAAGCUUGCG) ₂ (SEQ ID NO: 89) was constructed in Macromodel using PDB entry Ial5. Conformational optimization of neomycin, docked in the closest proximity for its 5' position to Hoechst-linker, was carried out prior to attachment to Hoechst using a Monte Carlo routine in MacroModel, using AMBER* force field and water as solvent. A similar protocol was used for Hoechst 33258. Five of the six amines in neomycin were protonated, in agreement with NMR studies of neomycin (Kaul, M., et al. J. MoI. Biol. 2003, 326. 1373-1387; Botto, R. E., et al. J. Am. Chem. Soc. 1983, 105, 1021-1028), as well as the terminal amine in the piperazine ring of the Hoechst moiety. Energy minimization reached a convergence threshold of 0.02 kJ/mol in all. cases. b) Results and Discussion

406. Neomycin — Hoechst 33258 conjugate significantly increases the thermal stability ofpolyA»polyURNA duplex. The thermal stability of polyA»polyU in the presence of neomycin, Hoechst 33258, and the neomycin-Hoechst 33258 conjugate (termed NHl) was first studied by thermal denaturation monitored by UV absorbance (Figure 35). Neomycin, as expected, was found to increase the T _m of the duplex, with a δT _m of 13 ⁰ C. Hoechst 33258 showed no increase in T _m . The conjugate, NHl, considerably precipitated the RNA, obvious to the naked eye as well as by observing the little change in UV absorbance upon sample heating. Therefore, relatively clean melting profiles were gathered by immediate UV melting analysis upon sample preparation. Close inspection of Figure 35 reveals a small amount of precipitation (small fluctuations in absorbance) even under these conditions. The resulting profiles, however, indicate a significant increase in the T _m , with a change of over 30 ⁰ C. Simple comparison of T _m values demonstrate that Hoechst binding is present in the conjugate. Interestingly, there is no observed stabilization by Hoechst 33258 alone, meaning that there is a requirement for neomycin's presence to effect any binding by the bis(benzimidazole) derivative. Therefore, both neomycin and Hoechst bind in tandem to significantly stabilize polyA»polyU, much more effectively than neomycin alone.

407. Circular dichroism of polyA»polyU in the presence of Neomycin — Hoechst 33258 conjugate indicates considerable conformational changes in both RNA and the Hoechst moiety. Circular dichroism (CD) spectroscopy was used to study the changes, if any, in the RNA duplex structure as well as in the Hoechst moiety of the conjugate. Numerous reports have explained binding — induced chirality of Hoechst 33258 by DNA, typically indicated by a change in CD signal at the λ _max of Hoechst 33258 UV absorbance (Rao, K. E., et al. Chem. Res. Toxicol. 1991, 4, 661-669; Canzonetta, C., et al. Bioctam. Biophys. Acta 2002, 1576, 136-142). Titrations of ligand into a solution of RNA were carried out with CD scans of the solution taken after appropriate equilibration times between ligand additions. The resulting scans were overlayed to compare the CD spectra at different ligand:RNA ratios (Figure 36a). There is a significant change in the CD signal in the polyA»polyU region, which is represented by a negative band at 245 nm and a positive band at 265 nm. Also, in the region of 345 nm, there is an increasingly negative CD signal as ligandrRNA ratios increased, indicative of Hoechst complexation with the RNA. Such a signal would be absent if no interaction is occurring. For neomycin titrations, significantly less change in CD signal is apparent. Therefore, these results further support Hoechst binding to the polyA»polyU duplex is occurring. As was observed with other experiments, no changes in the emission spectra occurred when Hoechst 33258 was mixed with polyA»polyU.

408. TheCD spectrum of the NHl - polyA*polyU complex was further evaluated by using CD melting experiments. These allow confirmation of T _m results obtained from UV melting studies, and the ability to monitor any conformational changes in a temperature dependent fashion. Since polyA«polyU is capable of forming complex structures when heated, it is likely these structures will be represented by changes in their CD spectra. Figure 37 clearly indicates a single melting transition of polyA«polyU alone. In the presence of ligand, CD changes were observed in the polyA»polyU region primarily occurred at temperatures around that in the absence of ligand (Figure 38b). However, when the CD values of the ligand - complex at increasing temperatures were compared, there was a clear transition at higher temperatures (similar to that observed in UV melting), confirming the melting of the complex and "freeing up" of the bound ligand (Figure 38c). These results strongly indicates that alternative structures are formed upon heating, likely attractive to the ligand since CD values were still virtually unchanged at temperatures where RNA duplex melting has occurred (as supported by the CD melting profile of the RNA region at 245 nm, Fig. 4b). Job analysis using

UV absorbance indicated RNA triplex formation at elevated temperatures, further validating this assertion.

409. Fluorescence titrations indicate Hoechst binding in the Neomycin — Hoechst 33258 conjugate to polyA»polyU. Numerous studies have used fluorescence spectroscopy to study Hoechst 33258 binding (Breusegem, S. Y., et al. Methods Enzymol. 2001, 340, 212-233; Loontiens, F. G., et al. Biochemistry 1990, 29, 9029-9039; Satz, A. L., et al. Bioorg. Med. Chem. Lett. 2000, 8, 1871-1880; Bostock-Smith, C. E., et al. Nucleic Acids Res. 1999, 27, 1619-1624).

Typically, upon interaction with DNA, the fluorescence of Hoechst 33258 is greatly enhanced due to binding. There are a limited number of accounts where Hoechst 33258 fluorescence changes (increases or decreases) in the presence of RNA, indicating ligand binding. Hoechst binding (in the conjugate NHl) to polyA»polyU was confirmed by conducting fluorescence titrations of both Hoechst 33258 and NHl with polyA»polyU. Similar to protocols found in the literature, a solution of ligand was titrated with a concentrated solution of nucleic acid. The resulting fluorescence emission spectrum was recorded after each titration and equilibration, and data from a single emission wavelength can be fit, depending on whether fluorescence saturation is reached and single binding sites, are present. The fluorescence of NHl was greatly enhanced upon additions of polyA»polyU (Figure 39), whereas in the case of Hoechst 33258, no change was observed. Therefore, the Hoechst moiety in the conjugate (NHl) binds polyA»polyU, likely in a similar mode to that of DNA. In the case of Hoechst 33258 alone, the absence of fluorescence change strongly indicates that no binding was occurring.

410. Isothermal titration calorimetry reveals identical stoichiometries of binding, as well as a greater than 10-fold increase in binding by the NHl conjugate when compared with neomycin. The intriguing results of UV and CD experiments prompted an investigation of the binding of NHl using isothermal titration calorimetry (ITC). The ITC experiments typically involved 10 μL injections of ligand solution into a sample chamber containing RNA, of which resulted in a heat burst curve for each injection, corresponding to the heat (given or taken) from the interaction between ligand and RNA. For Hoechst 33258 alone, little binding was observed, as indicated by heat burst curves similar to drug-into-buffer titrations. Neomycin titrated into duplex polyA.polyU displayed considerable binding (fit using Origin 5.0 software), with a binding constant of 1.2 x 10 ⁶ M ^"1 (Figure 40a). Using the same conditions for conjugate NHl (except for a lower ligand concentration due to early saturation in the isotherm due to stronger binding by NH 1 ), the binding constant K = I.6 x 10 ⁷ M ^"1 , a greater than 10-fold increase over neomycin alone binding to polyA«polyU (Figure 40b). Also, the δH of binding is 9 kcal lower

for NHl (-15.9 kcal/mol) than for neomycin (-6.9 kcal/mol). In both cases, the binding stoichiometrics are virtually the same, about 5 base pairs per ligand. These results provide support that the presence of the Hoechst moiety in the conjugate results in an increased affinity for the RNA duplex, and that the interaction likely involves both grooves of the duplex. Had the binding of the Hoechst moiety extended further up the polymeric lattice, the binding site size would be larger than neomycin. However, this was not the case. Also, it is unlikely that binding within the same groove is occurring, due to the size and composition of the linker between the two binding groups (as if they could fold together to bind a site of ~5 base pairs). Thus, dual groove binding by the conjugate is likely responsible for the observed signal in these experiments. All thermodynamic data gathered from ITC experiments are listed in Table 3. Table 3. ITC generated thermodynamic data for neomycin and NHl binding to polyApolyU duplex.

411. Viscometric titrations indicate groove binding instead of intercalation by the Hoechst moiety in the Neomycin - Hoechst 33258 conjugate. Given the substantial evidence by other experiments that Hoechst — RNA binding was indeed occurring, it was of great interest to substantiate the mode of binding using viscometry. Hoechst 33258 has been known to intercalate non - B - form nucleic acid structures (Moon, J.-H,, et al. Biopolymers 1996, 38, 593-606; Adhikary, A., et al. Nucleic Acids Res. 2003, 31, 2178-2186). Viscometry has long been utilized for investigating ligand - substrate binding modes, particularly to confirm intercalation events. Intercalation of nucleic acids results in an increase in the helical length, because of space displacement between the base pairs by the ligand. Nucleic acids of appropriate length are rod - like, so an increase in helical length results in an increase in solution viscosity (Cohen, G., et al. Biopolymers 1969, 8, 45-55). Thus, viscometric titrations of neomycin, Hoechst 33258, and NHl were carried out with polyA»polyU (Figure 41). Neomycin displayed characteristic strong groove binding, with a decrease in viscosity (most likely due to a compacting of the RNA structure, contrasting that of intercalation, which increases viscosity due to elongating of the rod-like nucleic acid structure). Such a decrease in viscosity has been observed before, particularly with aminoglycoside interactions (Arya, D. P., et al. J. Am. Chem. Soc. 2003, 125, 3733-3744; Jin, E., et al. J. MoI. Biol. 2000, 298, 95-110). Hoechst 33258, in accordance with other experiments, showed no change in the polyA»polyU solution viscosity.

NHl displayed even more decrease in solution viscosity when compared to neomycin, indicating stronger groove binding and shortening of RNA. Being the standard seal for determining intercalation interactions, these viscometry results indicate that binding of the Hoechst 33258 moiety is not of an intercalative nature. Furthermore, both ITC and CD titrations indicate identical stoichiometries of interaction (neomycin vs. NHl). Since aminoglycosides are widely considered to bind the major groove of RNA, these results support a strong candidacy for a dual recognition agent in NHl, with interactions covering both major and minor groove of duplex RNA.

412. The Neomycin — Hoechst 33258 conjugate significantly stabilizes shorter RNA duplexes. To test whether or not the neomycin-Hoechst 33258 conjugate could bind and stabilize smaller sequences of RNA, self-complementary duplexes r(CGC AAAUUUGCG) ₂ (SEQ ID NO:881) and r(CGCAAGCUUGCG) ₂ (SEQ ID NO:89) were utilized. Due to the enhanced stabilization of d(CGCAAATTTGCG) ₂ (SEQ E) NO:90) by NHl over Hoechst 33258, it was exciting to ponder whether the conjugate (NHl) would display enhanced stabilization of the RNA analog. Furthermore, the requirement for an A/U stretch was also probed by carrying out comparison experiments with a GC junction in place of the centra! AU junction of r(CGC AAAUUUGCG) ₂ (SEQ ID NO:88).

413. For a clear comparison of each ligand's effect on the thermal stability, samples of r(CGCAAAUUUGCG) ₂ (SEQ ID NO:88) were prepared individually in the presence of neomycin, Hoechst 33258, and NHl. Also, a solution of r(CGC AAAUUUGCG) ₂ (SEQ ID NO:88) in the presence of both neomycin and Hoechst 33258 was prepared. Such a comparison could indicate whether neomycin binding induces a conformational preference in the duplex for Hoechst 33258 to bind. This indeed was not the case. We found that NHl significantly enhances the T _n , of the RNA duplex (δT _m = 11 ⁰ C, Figure 42). Neomycin displayed a slightly less shift in the T _m (δT _m = 4 ⁰ C), whereas Hoechst 33258 slightly destabilized the duplex T _m . The mixture of neomycin and Hoechst 33258 resulted in a T _m in between those obtained with neomycin and Hoechst 33258 alone. Additional UV melting experiments of NHl and neomycin with r(CGCAAGCUUGCG) ₂ (SEQ ID NO:89) indicated a slight stabilizing effect by NH-I, though not much greater than neomycin alone (2 degrees higher). Therefore, the conjugation of Hoechst 33258 with neomycin is necessary for Hoechst binding, and the binding for A _n U _n stretches are significantly more favorable than G/C containing sequences.

414. Fluorescence titration experiments further corroborated a sequence specificity of NHl for the A ₃ U ₃ stretch (Figure 43). As with poly(A)»poly(U), fluorescence of NH-I is

enhanced upon mixing with the 12mer RNA duplexes. Fluorescence mixing curves (Job plots) indicated clear 1:1 binding for NHl in both r(CGCAAAUUUGCG) ₂ (SEQ ID NO:88) and r(CGCAAGCUUGCG) ₂ (SEQ ID NO:89). Therefore, equilibrium titrations of RNA into NHl could be fit to a one site binding model to give K _b values of 6.5+1.6 x 10 ⁶ M ^"1 and 1.2+0.3 x 10 ⁶ M- ¹ for r(CGCAAAUUUGCG) ₂ (SEQ ID NO:88) and r(CGCAAGCUUGCG) ₂ (SEQ ID NO:89), respectively. As expected, Hoechst 33258 alone displayed no fluorescence increase upon mixing with RNA. Moreover, the fluorescence was observed to slightly decrease, as illustrated in a fluorescence Job plot of Hoechst 33258 with r(CGCAAAUUUGCG) ₂ (SEQ ID NO:88).

415. These results further illustrate that NHl is not only a potent DNA duplex binding molecule, but a potent RNA duplex binding molecule as well. Furthermore, a specificity of Hoechst for AAJ base pairs exists, potentially due to a similar phenomenon that explains A/T base specificity in DNA.

416. Molecular Basis for Duplex Recognition by Hoechst 33258 and Neomycin- Hoechst 33258 conjugate (NHl). RNA duplexes adopt an A-form conformation. The A-form family of nucleic acids consist of right-handed, antiparallel double helices which possess a shallow, but wide, minor groove and a deep, narrow major groove, which are largely the result of the approximately 4 angstrom displacement of the base pairs. This largely contrasts with B- DNA, which maintains a narrow, deep minor groove and a wide, shallow major groove. The number of base pairs per helical turn is 11 for A-DNA, whereas B-DNA maintains one less per turn. The ~30° reduction in helical twist is also a characteristic of A-form when comparing to B- form. The rise per base pair can be nearly one angstrom less than B-DNA, and the base pair inclination is much greater in A-form (between approximately 10 ° — 20 °, compared to 0 ° for B- DNA. Typically, B-form DNA is associated with sugar puckering of C2'-endo, while A-form consists of C3'-endo conformation, which give rise to ~1 angstrom difference in the phosphate- phosphate separations between each conformation. The C3'-endo is the more stable conformation due to the presence of the C2' - hydroxyl.

417. Hoechst 33258 binding: DNA vs. RNA. The driving force of the Hoechst — nucleic acid interaction is isohelicity (Goodsell, D., et al. J. Med. Chem. 1986, 29, 727-733). Like distamycin and netropsin - based ligands, the crescent molecular shape of bis(benzimidazoles) such as Hoechst 33258 matches well the pitch of the DNA minor groove. The forces driving the interaction are a combination of van der Waals interactions, hydrogen - bonding, and electrostatic (ammonium group of the terminal piperazine ring). Hydrogen bonding has typically

been observed between the imidazole NH and either thymine 02 or adenine N3 positions within A/T base pair stretches (Vega, M. C ₃ et al. Eur. J. Biochem. 1994, 222, 721-726; Teng, M. K., et al. Nucleic Acids Res. 1988, 16, 2671-2690; Spink, N., et al. Nucleic Acids Res. 1994, 22, 1607- 1612; Searle, M. S., et al. Nucleic Acids Res. 1990, 18, 3753-3762; Pjura, P. E., et al. J. MoI. Biol. 1987, 197, 257-271; Harshman, K. D., et al. Nucleic Acids Res. 1985, 13, 4825-4835). Molecular modeling of Hoechst 33258 with d(CGCAAATTTGCG) ₂ indicated such interactions (Figure 44). The recognition of DNA, however, does not rely solely on the number and type of hydrogen bonding contacts. Numerous studies have indicated that the sequence specificity of such crescent shaped ligands is dominated by van der Waals interactions between the ligand the groove walls and floor (Nunn, C. M., et al. J. Med. Chem. 1995, 38, 2317-2325; Kopka, M. L., et al. Proc. Nat. Acad. Sci. U.S.A. 1985, 82, 1376-1380; Czarny, A., et al. J. Am. Chem. Soc. 1995, 117, 4716-4717).

418. Molecular modeling of Hoechst 33258 docked in the minor groove of RNA analog r(CGC AAAUUUGCG) ₂ , as with DNA, indicates a number of hydrogen bonding interactions can potentially occur (Figure 45). Yet experimental evidence indicates that no significant interactions between Hoechst 33258 and RNA duplex (polymeric and oligomeric) occur (our work and others) (McConnaughie, A. W., et al. J. Med. Chem. 1994, 37, 1063-1069).

RNA, adopting the A-form conformation, possesses a minor groove much wider than that observed for B-DNA. Therefore the tight fit, that occurs with ligands such as Hoechst 33258 and B-DNA, is absent in RNA duplex. Nonetheless, Hoechst 33258 has been shown to bind bulge and loop - containing RNA duplexes, primarily the bubble regions (Dassonneville, L., et al. Nucleic Acids Res. 1997, 25, 4487-4492; Cho, J., et al. Nucleic Acids Res. 2000, 28, 2158- 2163; Dominick, P., et al. Bioorg. Med. Chem. Lett. 2004, 14, 3467-3471). Little has been investigated as to the molecular contacts, so geometrical considerations are general at the least. One can, however, deduce that H — bonding can play a role in this recognition, given the number of H — bonding sites in the ligand and the absence of geometry similar to the B-DNA minor groove that encourages favorable van der Waals interactions.

419. Similar to Hoechst 33258 alone, the Hoechst moiety in NHl can be shown to display similar contacts with the DNA minor groove (Figure 46). Close inspection of the molecular model of the NHl-d(CGCAAATTTGCG) ₂ complex indicates a fit between the benzimidazoles region and groove walls similar to that of Hoechst 33258. The neomycin moiety is observed to also display a number of hydrogen bonding interactions within the major groove. The dual nature of binding portrayed by the conjugate is supported experimentally with

observations that enhanced stabilization of DNA occurs when compared with Hoechst 33258 alone (Arya, D. P., et al. J. Am. Chem. Soc. 2003, 125, 12398-12399). Furthermore, the major groove interactions may explain previous observations that third strand binding by a triplex forming oligonucleotide in the duplex major groove (to form a DNA triplex) is not observed under normal triplex - forming conditions with the conjugate (NHl) present (Arya, D. P., et al. J. Am. Chem. Soc. 2003, 125, 12398-12399).

420. Contrasting DNA and small molecules that are limited to binding its minor groove, small molecules known for binding RNA are cationic in nature (e.g., aminoglycosides such as neomycin) and thus bind in the RNA major groove. The larger negative potential (Saenger, W. Principles of Nucleic Acid Structure; Springer-Verlag: New York, 1984) in the major groove draws more attention than the minor groove in RNA. Moreover, a number of groups have established aminoglycosides as binding within the RNA major groove (Fourmy, D., et al. J. MoI. Biol. 1998, 277, 347-362; Jin, E., et al. J. MoI. Biol. 2000, 298, 95-110; Carter, A. P., et al. Nature 2000, 407, 340-348). Li the present research, we have modeled NHl for binding r(CGCAAAUUUGCG) ₂ , with neomycin docked within the major groove and the Hoechst region rested within the minor groove of the central A ₃ U ₃ stretch. The hydrogen bonding pattern of the Hoechst region in NHl with the RNA duplex is similar to the corresponding DNA d(CGCAAATTTGCG) ₂ (Figure 47). Yet the snugness of fit within the minor groove does not impress like that with the DNA minor groove. The observed interaction is likely driven by the neomycin moiety. This is supported by equilibrium binding data indicating binding constants in the 10 ⁶ (M ^"1 ) range in RNA binding (observed for neomycin in ITC experiments), contrasting that of Hoechst 33258 with DNA, which renders binding constants two orders of magnitude greater. Furthermore, the lack of favorable van der Waals interactions is conceivable by observing the low quantum yield in fluorescence measurements when compared with DNA. Had DNA-like groove interactions occurred, fluorescence due to Hoechst binding should be more enhanced due to diminished solvent exposure.

421. DNA binding: Hoechst 33258 vs. NHl. A comparison of Hoechst 33258 vs. NHl minor groove interactions can be made by observing the H-bonding interactions depicted in Figures 45 and 46. In viewing the model of the NHl - DNA complex, the substituted phenol region of the Hoechst moiety appears somewhat tugged out of the groove, possibly due to neomycin exhibiting favorable interactions within the major groove. Also, the H-bonding distances for NH-I are slightly larger than that observed with Hoechst 33258 alone. Experimental evidence supports an increased stabilization of DNA duplex by NHl over Hoechst

33258 alone, indicating neomycin's role in binding (Arya, D. P., et al. J. Am. Chem. Soc. 2003, 125, 12398-12399). Though the snug fit within the minor groove is somewhat diminished in NHl, the favorable contacts of neomycin counteract the discreet loss in optimal binding geometry of the Hoechst moiety.

422. RNA binding: Hoechst 33258 vs. NHl. Hoechst 33258 binding to the minor groove of RNA seems probable in the current modeling studies. Similar H-bonding contacts are observed both with the Hoechst moiety of NHl and with DNA. However, various experiments clearly convey that the RNA duplex binding effect elicited by the ligand is minimal, if at all. In agreement with reported accounts of isohelicity dominating the high binding affinity and specificity, Hoechst 33258 simply does not optimally match the pitch of the RNA minor groove, and therefore van der Waals contacts otherwise present in DNA interactions are significantly diminished. If one were to consider the modeling of Hoechst 33258 with RNA to be fully reliable, the fact that H-bonding interactions are displayed between Hoechst 33258 and RNA, then the postulate that van der Waals interactions dominate binding is further supported by the presented research.

423. Various techniques described in this paper support, by conjugation of a Hoechst 33258 - based derivative with neomycin, favorable interactions between Hoechst and the RNA minor groove, a phenomenon unobserved with Hoechst 33258 alone. The conjugate, with neomycin binding in the major groove, places the Hoechst moiety in good proximity to the minor groove, where the theoretical H-bonding interactions could be a reality.

424. Sequence Specificity of Hoechst 33258 for DNA. Numerous accounts have found that the recognition of DNA minor groove by small molecules relies on the van der Waals interactions between the ligand and 2'-deoxyribose walls of the minor groove (Nunn, C. M., et al. J. Med. Chem. 1995, 38, 2317-2325; Kopka, M. L., et al. Proc. Nat. Acad. Sci. U.S.A. 1985, 82, 1376-1380; Czarny, A., et al. J. Am. Chem. Soc. 1995, 117, 4716-4717). The primary sites largely involve A/T sequences, which are narrower than G/C base pairs. The H-bonding patterns that occur are considered a consequence of localization of the ligand within the groove. Dervan's rules for base pair recognition (White, S., et al. Nature 1998, 391, 468-471), specifically G/C base pairs, rely on the avoidance of steric hindrance between the 3 position C-H in pyrroles and the 2-amino of guanine by utilizing imidazole groups, whose nitrogen can act as H-bond acceptors (Kopka, M. L., et al. Proc. Nat. Acad. Sci. U.S.A. 1985, 82, 1376-1380). Therefore, recognition by hydrogen bonds signify a role in the sequence recognition process, albeit a consequence of the snug fit of the crescent shaped distamycin - based ligands to the

width and pitch of the minor groove. Hoechst 33258 has been shown on multiple occasions to bind a A/T stretches of 4-5 base pairs (Vega, M. C, et al. Eur. J. Biochem. 1994, 222, 721-726; Teng, M. K., et al. Nucleic Acids Res. 1988, 16, 2671-2690; Spink, N., et al. Nucleic Acids Res. 1994, 22, 1607-1612; Searle, M. S., et al. Nucleic Acids Res. 1990, 18, 3753-3762; Pjura, P. E., et al. J. MoI. Biol. 1987, 197, 257-271; Harshman, K. D., et al. Nucleic Acids Res. 1985, 13, 4825-4835). Disruption of the stretch with a G-C pair significantly alters the binding, a consequence of the unfavorable steric clash between the guanine NH ₂ (at the 2 position) and the N-H of the imidazole.

425. The preference for the A ₃ U ₃ stretch in the RNA duplex. r(CGCAAGCUUGCG) ₂ is also observed in a comparison with r(CGCAAGCUUGCG) ₂ . The higher binding affinity (Figure 43) may be due to steric clash between imidazole NH and the 2-amino group in guanine. Upon replacement of the central AU junction in the A ₃ U ₃ RNA with GC, molecular modeling of the RNA complexed with NHl, upon minimization, indicates a -30° rotation of the benzimidazole on the piperazine side. The benzimidazole on the phenol side does not indicate significant rotation, likely because of the proximity of the guanine NH ₂ to the imidazole, which is pulled slightly out of the groove by neomycin's multiple contacts in the major groove. Consequently, fluorescence studies show that binding is nearly 6 times greater when the central GC is replaced with AU. The observation that binding is still great (K ~10 ⁶ ) with the A ₂ GCU ₂ duplex can be attributed to interactions imposed by neomycin, an infidel in RNA binding (Arya, D. P., et al. J. Am. Chem. Soc. 2003, 125, 10148-10149).

4. Example 5: Synthesis of Neomycin-DNA/Peptide Nucleic Acid Conjugates a) Materials and Methods

426. AU commercial reagents were used without further purification. Pyridine, methylene chloride, and N,7V-dimethylformamide (DMF) were refluxed over calcium hydride and distilled. All reactions were carried out in oven-dried glassware under νi/argon atmosphere. AU reactions were monitored by thin-layer chromatography (TLC) with pre-coated silica gel on glass plates. TLC plates were visualized with either UV light or staining solutions such asp- anisaldehyde or phosphomolybdic acid, followed by heating the plate with a heating gun. ICν silica gel 32-63 (60 A) was used for column chromatography. ¹ H νMR spectra were recorded either using a 300 MHz or a 500 MHz νMR. JR was recorded on a νicolet Magna-IR™ sρectrometer-550 either as a solution of 1,2-dichloroethane or CCl ₄ and the peaks corresponding to the solvent was subtracted manually. MALDI-TOF mass was recorded on a Bruker Daltonics

OmniFLEX™ Bench-Top MALDI-TOF mass spectrometer. Preparative anion exchange HPLC was carried out on a Phenomenex SAX 80 A column (10x250 mm, 5μ) and for analytical anion exchange HPLC, a Waters Gen-Pak FAX (4.6x100 mm) column was used. Preparative and analytical RP-HPLC was carried out with Alltima C8 100 A (250x4.6mm, lOμ) column.

(1) Preparation of neomycin isothiocyanate 3

427. To a stirred solution of neomycin amine 2 (Figure 53) (60mg, 0.05mmol) in anhydrous CH ₂ Cl ₂ was added l,r-thiocarbonyldi-2(lH)pyridone (12mg, 0.05mmol) and DMAP (lmg, 0.008mmol) under an argon atmosphere. After 13 h, the solution was concentrated and loaded on a silica gel column. Flash chromatography with EtOAc as the eluent yielded neomycin isothiocyanate 3 (Figure 53) in 48% yield (30mg) ; R _f = 0.55 (10% CH ₃ OH in CH ₂ Cl ₂ ); ¹ HNMR (300MHz, CDCl ₃ ) δ ppm : 1.4 (m, 54H), 1.89 (2H), 2.85-2.9 (4H), 3.0-3.3 (9H), 3.40-3.52 (6H), 3.76 (2H), 3.89 (2H), 4.15(2H), 4.23 (IH), 4.92 (IH), 5.13 (IH), 5.3 (br, IH); IR (as a solution in CCl ₄ followed by subtraction of solvent peaks) v cm ^"1 ; 1160.71, 1270.95, 1421.9, 1511.7, 1692.44 (>CO stretch), 2305.5 (>N=C=S stretch), 2986.74, 3045.28. Maldi MS: 1315.60, found 1334.55 (M+H ₂ 0).

(2) Covalent attachment of neomycin to dTiβ

428. Oligonucleotide synthesis was carried out on an Expedite Nucleic Acid Synthesis System (8909) using standard phosphoramidite chemistry. 5'-amino-5'-deoxythymidine 5 (Figure 55) was synthesized from thymidine using reported procedures (Bannwarth, W. Helvetica Chimica Acta 1988, 71, 1517-1527; Tetzlaff, C. N., et al. Tetrahedron Letters 1998, 39, 4215- 4218). The 3 '-phosphoramidite of 5'-amino-5'-deoxythymidine was added to the 5'-end of the dTj ₅ (7, Figure 56) with a 20 min coupling time.

429. After oligomer synthesis, the CPG column was dried using argon gas. A syringe containing 0.1 M pyridine solution of neomycin isothiocyanate 3 (Figure 53) and 10 mol% of DMAP was attached to one end of the CPG column containing oligomer 8 (Figure 56) and another end was attached to an empty syringe. The solution was transferred from one syringe to another and left undisturbed for 30 min. After 30 minutes, the solution was pushed back to the other syringe and this push and pull procedure was repeated every 30 min. After 12 h, the column was detached from both syringes, washed with 5x1 ml OfCH ₂ Cl ₂ and dried by flushing with argon gas. Conjugate 9 (Figure 56) was then detached from the solid support using NH ₄ OH and the resulting solution was evaporated. RP-HPLC purification using triethylammonium acetate buffer yielded pure conjugate 9 (Figure 56). The dried sample was treated with 1 ml of 1,4-dioxane solution containing 5% CF ₃ CO ₂ H and 1% w-cresol (v/v/v %). After 30 min,

conjugate 10 (Figure 56) was precipitated with 10 ml of diethyl ether and the precipitate was washed with 3x 10 ml of diethyl ether.

430. Conjugate 10 (Figure 56) was purified by preparative anion exchange HPLC using a tris buffer system (buffer A: 25 mM of tris.HCl and 1 mM of EDTA, buffer B; buffer A and 1 M NaCl; 0-60% of buffer B over buffer A during 60 min). The fractions were collected and concentrated. The major fractions were checked for their identity using MALDI-TOF mass spectrometry.

(3) Covalent attachment of neomycin to Tio PNA oligomer 11

431. PNA oligomer synthesis was carried out on Expedite Nucleic Synthesis System (8909) using standard PNA chemistry on a PAL resin {5-(4'-Aminomethyl-3',5'- dimethoxyphenoxy)-valeric acid resin}. At the end of the synthesis, Fmoc-free 5'-amino group containing PNA oligomer 11 (Figure 59) was dried by flushing with argon gas. A portion of the sample was removed and deprotected from the column using the following procedure. A syringe containing a solution OfCF ₃ CO ₂ H (TFA) and w-cresol (4:1, v/v%) was attached to one end of the PAL resin column and another end was attached to an empty syringe. The solution was. transferred from one syringe to another and left undisturbed for 30 minutes. After 30 minutes, the solution was transferred back to the other syringe and this push and pull procedure was repeated every 30 minutes. After 90 minutes, the syringes were detached from the column and the column was rinsed with 5x0.3 ml of TFA. T ₁₀ PNA 11 (Figure 59) was then precipitated with ethyl ether and centrifuged. The precipitate was washed with ethyl ether and dried. A portion of T ₁₀ PNA 11 (Figure 59) was stirred with a pyridine solution containing 0.1 M solution of neomycin isothiocyanate 3 (Figure 53) and 10% of DMAP. After 12 h, the conjugate was precipitated with 10 ml of diethyl ether and the precipitate was further washed with 3x10 ml of diethyl ether. The Boc groups on neomycin were deprotected with IM HCl/dioxane in the presence of 1,2-ethanedithiol to give 12 (Figure 59).

432. Conjugate 12 (Figure 59) was purified by preparative PvP-HPLC using a TFA buffer system (buffer A: 0.1 % of TFA in water, buffer B: 0.1% TFA in acetonitrile). The fractions were collected, concentrated, and pooled together after checking their identity using HPLC and MALDI-TOF mass spectrometry. b) Results and Discussion

(1) Covalent attachment of neomycin to a DNA oligomer

433. The amino groups on rings I, II and IV (neomycin, Figure 1, Figure 52) are necessary in stabilizing and recognizing various nucleic acid forms (aminoglycosides without

any of these amines do not stabilize rRNA, DNA/RNA triplexes as efficiently) (Arya, D. P., et al. J. Amer. Chem. Soc. 2001, 123, 5385-5395; Arya, D. P., et al. Bioorg Med Chem Lett 2000, 10, 1897-9; Arya, D. P., et al. J. Amer. Chem. Soc. 2001, 123, 11093-11094; Charles, L, et al. Bioorg Med Chem Lett 2002, 12, 1259-62; Xue, L., et al. J. Chem. Soc. Chem. Commun. 2002, 70-71). The conjugates based on aminoglycosides must then retain these amines. The 5"-OH on the ribose of ring EI (neomycin) was thus chosen to provide the linkage to the nucleic acids. The key synthetic step involves coupling of neomycin isothiocyanate with 5' -amino group of the modified DNA to give the isothiourea linkage (Figure 52).

434. Synthesis of neomycin isothiocyanate (Charles, L, et al. Bioorg Med Chem Lett 2002, 12, 1259-62) 3 (Figure 53) was initiated by conversion of the natural product-neomycin to 5"-deoxy-5"-amino neomycin 2 (Figure 53). Reaction of this amine and l,r-thiocarbonyldi- 2(lH)pyridone (TCDP) in the presence of 4-N,iV-dimethylaminopyridine (DMAP) leads to the formation of isothiocyanate, which can be easily followed by IR spectroscopy (Figure 54).

435. The IR of neomycin isothiocyanate shows the appearance of a new peak at l/λ=2305.50 cm ^"1 , which corresponds to the isothiocyanate group.

436. The DNA oligomer was modified at the 5'-end by introducing 5'-amino-5'- deoxythymidine 5 (Figure 55) at the end of the oligomer synthesis. The synthesis of 5'-amino-5'- deoxythymidine 5 from thymidine 4 is described in Figure 55. The amino group was protected with 4-methoxyphenyldiphenylmethyl (MmTr) group, which was followed by phosphitylation using standard phosphoramidite chemistry to get 6 (Figure 55) in good yields.

437. The synthesis of dT ₁₅ 7 (Figure 56) was carried out on a control pore glass (CPG) column using standard phosphoramidite chemistry. After oligomer synthesis, the άi(p- methoxyphenyl) phenylmethyl (DMTr) group on the 5'-hydroxyl group of dT ₁₅ 7 was deprotected with 4% CC1 ₃ CO ₂ H/CH ₂ C1 ₂ (Figure 56). MmTr-protected 5'-amino-5'- deoxythymidine phosphoramidite 6 was coupled with the 5'-hydroxyl group of dT ₁₅ 7 in the presence of lH-tetrazole as a coupling reagent with extended coupling time (20 min). After oxidation and capping steps, 5'-MmTr group was deprotected with 4% CC1 ₃ CO ₂ η/Cη ₂ C1 ₂ to give 8 (Figure 56). The CPG column containing the oligomer 8 (Figure 56) was then dried with argon gas.

438. The 5'-amino group of the oligomer was reacted with a 0.1 M solution of neomycin isothiocyanate 3 and 10 mol% DMAP. After 12 h, the column was washed with 5xlml OfCH ₂ Cl ₂ and dried by flushing with argon gas. The conjugate 9 (Figure 56) was then

detached from the solid support using NH4OH and purified by preparative reverse phase HPLC (RP-HPLC) using a triethylammonium acetate buffer system. The dried sample was treated with a 1,4-dioxane solution containing 5% CF ₃ CO ₂ H and 1% m-cresol (v/v/v %). After 30 min, the deprotected conjugate 10 (Figure 56) was precipitated and washed with excess diethyl ether. The deprotected conjugate 10 was finally purified by preparative anion exchange HPLC using a Tris.HCl buffer system (Figure 57).

439. Conjugate 10 (Figure 56) elutes with a retention time of 6.07 min, whereas the nonconjugated dT ₁₆ elutes at 7.22 min. All major fractions were pooled together and checked for their identity using MALDI-TOF mass spectrometry (Figure 58). The expected mass for neomycin-DNA conjugate is m/z 6202.30 Da and the MALDI-TOF mass spectrum showed a peak at m/z 6202.90 Da, confirming the identity of the desired compound.

(2) Covalent attachment of aminoglycoside to PNA oligomer

440. The 5'-amino group of T ₁₀ PNA 11 (Figure 59) was similarly reacted with neomycin isothiocyanate in the presence of DMAP and pyridine to give PNA-neomycin conjugate connected through a thiourea linkage. T ₁₀ PNA 11 (Figure 59) was first synthesized using the standard PNA chemistry protocols and deprotected from solid support.

441. After precipitation, the sample was dried and stirred with a pyridine solution containing neomycin isothiocyanate and DMAP for 12 h. The solution was evaporated and washed with diethyl ether. Boc groups on neomycin were deprotected with trifluoroacetic acid/m-cresol (4:1) in methylene chloride (v/v%). Conjugate 12 (Figure 59) was then purified with RP-HPLC using a trifluoroacetic acid buffer system (Figure 60).

442. HPLC purification and characterization of T ₁₀ PNA 11 (Figure 60) has been found to be difficult because of poor solubility in water and self-aggregation even at lower PNA concentrations (50 μM). After introducing one lysine residue at the 3 '-end, T ₁₀ PNA solubility increases and self-aggregation also decreases (Egholm, M., et al. J. Amer. Chem. Soc. 1992, 114, 1895-1897; Tackett A. J., et al. Nucleic Acids Res. 2002, 30, 950-7; Corey, D. J. TIBTECH 1997, 15, 224-229; Lesnik, E., et al. Nucleosides & Nucleotides 1997, 16, 1775-1779; Gildea, B. D., et al. In PCT Int. Appl.; (Boston Probes, Inc., USA). Wo, 1999, p 81 pp; Gildea, B. D., et al. Tetrahedron Letters 1998, 39, 7255-7258). Conjugation OfT ₁₀ PNA with neomycin remarkably increased the solubility and conjugate 12 (Figure 59) did not self-aggregate even at high PNA concentrations.

443. Table 4. HPLC retention times of PNA and PNA-neomycin conjugates. Buffer conditions: buffer A: 0.1% of TFA in water; buffer B: 0.1% of TFA in acetonitrile; for PNA, 0-

100% buffer B during 7 min; 50 °C; for PNA conjugate, 0-30% of buffer B over buffer A during 13 min; 30-100% buffer B during 2 min, 65 °C.

444. This is, most likely, due to the introduction of six amino groups in the conjugate. The identity of the molecule was confirmed with MALDI-TOF (Figure 61). The mass spectrum of the conjugate 12 (Figure 59) showed a molecular ion peak at m/z 3398.37 Da (calculated m/z - 3398.22 Da).

445. A few other PNA sequences which are complementary to biologically important DNA/RNA sequences were prepared and their neomycin conjugates were also successfully prepared (Praseuth, D., et al. Biochim. Biophys. Acta 1996, 1309, 226-238; Mologni, L., et at . Biochem Biophys Res Commun 1999, 264, 537-43; Armitage, B., et al. Nucleic Acids Resl998, 26, 715-720; Good, L., et al. Proc Natl Acad Sci U S A 1998, 95, 2073-2076; Kurg, R., et al. Virus Research 2000, 66, S9-50-, Mologni, L., et al. Nucleic Acids Res 1998, 26, 1934-8). Their synthesis and purification were carried out in a manner similar to that of the PNA T _ϊ o-neomycin conjugate 12 (Figure 59). The HPLC retention times of these PNAs with and without neomycin conjugation are listed in Table 4.

5. Example 6: Sequence-Specific Targeting of RNA with an Oligonucleotide-Neomycin Conjugate a) Materials and Methods

446. AU commercial reagents were used without further purification. Pyridine, methylene chloride and λζiV-dimethylformamide (DMF) were refluxed over calcium hydride and distilled. All reactions were carried out in oven-dried glassware under N ₂ /argon atmosphere. AU reactions were monitored by thin-layer chromatography (TLC) with pre-coated silica gel on glass plate. Visualization of the TLC plate was achieved with either UV light or spraying ethanolic ninhydrin solution followed by heating the plate with heating gun. ICN silica gel 32-63 (6θA) was used for column chromatography. ¹ H NMR spectra were recorded either using 300 MHz or 500 MHz NMR. IR was recorded on a Nicolet Magna-IR™ spectrometer-550 either as a solution of 1,2-dichloroethane or CCl ₄ and the peaks corresponding to the solvent was subtracted

manually. MALDI-TOF mass was recorded on a Bruker Daltonics OmniFLEX™ Bench-Top MALDI-TOF mass spectrometer. Preparative anion exchange HPLC was carried out on a Phenomenex SAX 80 A column (10x250 mm, 5μ) and for analytical anion exchange HPLC, a Waters Gen-Pak FAX (4.6x100 mm) column was used. Preparative and analytical RP-HPLC were carried out with Alltima C8 100 A (250x4.6mm, lOμ) column.

447. 7mer DNAs (dY, N-dY and dY' ) were synthesized on Expedite Nucleic Acid System (8909) and purified by HPLC. RNA (rR) was purchased from Dharmacon, Inc., (Lafayette, CO) and was used without further purification [lot numbers COFRA-0001 and CHAIB-OOOl]. The concentrations of all the nucleic acid solutions were determined spectrophotometrically using the following extinction coefficients (on a per strand basis, M ^-1 Cm ^{" l} ): ε ₂₆₀ = 81,900 for rR, and ε ₂₆₀ = 54,000 for dY. Neomycin (lot 129H0918) was purchased from Sigma (St. Louis, MO) and was used without further purification.

(1) UV Spectroscopy

448. UV spectra were recorded at λ = 200-300 ran on a Gary IE UV/Vis spectrophotometer equipped with temperature programniing. Spectrophotometer stability and λ alignment were checked prior to initiation of each melting point experiment. For accurate T _n , determinations, first derivative functions were used. Data were recorded every 1.0 degree. In all UV experiments, the samples were heated from 20 - 95 ⁰ C at a rate of 5 deg/min, annealed (95 - 5°C, at a rate of 0.2 deg/min rate) and again melted (5 - 90°C, at a rate of 0.2 deg/min rate) and the samples were brought back to 25°C at a rate of 5°C/min. During melting and annealing, the absorbance of each solution was monitored at the following wavelengths; 260nm, 280nm and 284nm. The second melting stage was used for calculating melting temperature.

(2) CD Spectroscopy

449. AU CD experiments were conducted at 15°C on a JASCO J-810 spectropolarimeter equipped with a thermoelectrically controlled cell holder. A quartz cell with a 1 cm path length was used in the CD studies. CD spectra were recorded as an average of 3 scans from 220 to 320 nm with data recorded in 1 nm increments with an averaging time of 2 s. The hybrid duplex solution was incubated at 4°C for 16 h before CD titration. After each addition of 5μl of neomycin (ImM) to a 2 ml solution of hybrid duplex (lOμM per duplex), the solution was inverted three times to ensure thorough mixing and incubated for 3 min prior to scanning. Buffer conditions for CD titration are: 10 mM sodium cacodylate, 0.5 mM EDTA, 60 mM total Na ⁺ , and pH 6.0.

(3) ITC Measurements

450. Isothermal titration calorimetric measurements were performed at 10 and 15 ⁰ C on a MicroCal VP-ITC (MicroCal, Inc.; Northampton, MA). In a typical experiment, 5μL aliquots of 500μM drug were injected from a rotating syringe (300 rpm) into an isothermal sample chamber containing 1.42 mL of a duplex solution that was 20μM per duplex. Each experiment was accompanied by control experiments in which 5μL aliquots of the drug was titrated into buffer alone. The duration of each injection was 5.0 s and the delay between each injection was 300 s. The initial delay prior to the first injection of 2μL was 60 s. Each injection generated a heat burst curve (microcalories per second vs. seconds). The area under each curve was determined by integration using the Origin 5.0 software (MicroCal, Inc.; Northampton, MA) to obtain a measure of the heat associated with that injection. The buffer used in the experiment was 10 mM sodium cacodylate, 0.5 mM EDTA, 60 mM total Na ⁺ , and pH 6.0.

(4) Preparation of conjugate 3:

451. To a stirred solution of neomycin isothiocyanate 2 (90 mg, 0.072 mmol) in pyridine was added amino deoxyuridine 1 (Kahl, J. D., et al. J. Amer. Chem. Soc. 1999, 121, 597-604) (38.5 mg, 0.072 mmol) and DMAP (3 mg, 0.008 mmol) under argon atmosphere. The reaction was carefully protected from exposure to light. After 22 hr, the reaction mixture showed the absence of amino deoxyuridine. The reaction mixture was concentrated and loaded on a silica gel column. Flash chromatography with 0-10% of methanol gave the required product as a white solid (100 mg, 76%).; IR (recorded on KBr pellet) v cm ⁴ ; 1050, 1180, 1261, 1450, 1695(>C=O stretching), 2991, 3392(O-H stretching); ¹ HNMR (500 MHz, DMSO-D6): δ ppm 0.04 (s, 3H), 0.05 (s, 3H), 0.10 (s, 3H), 0.11 (s, 3H), 0.86 (s, 9H), 0.91 (s, 9H), 1.25 (m, 2H), 1.4 (m, 54H), 1.64 (m, 6H), 1.99 (m, IH), 2.10 (br, 2H), 2.27 (m, IH), 2.38 (t, 2H, J= 6.3 Hz), 3.1-3.3 (m, 4H), 3.0-3.9 (m, 13H), 4.1 l(m, 2H), 4.15-4.40 (m, 4H), 4.41-4.50 (m, IH), 4.55-4.7 (m, 2H), 4.82 (m, 2H), 5.01 (m, IH), 5.15 (m, IH), 5.3 (m, 2H); 5.48 (s, 2H), 6.26 (t, IH ₅ J= 6.3 Hz) ₅ 7.02 (s, IH), 7.68 (s, IH);

(5) Preparation of acetylated conjugate 4

452. To an ice cooled solution of the conjugate 3 (75 mg, 0.042 mmol) in pyridine was added trifluoroacetyl anhydride (0.087ml, 0.42 mmol) and stirred 10 minutes and brought to room temperature and stirred for Ih. The solution was evaporated, redissolved in 100 ml of EtOAc, washed with 2 x 20 ml of brine solution, dried over Na ₂ SO ₄ , filtered, and concentrated under vacuum. Flash chromatography gave the required compound (74mg, 75%). IR (recorded

on KBr pellet) v cm ^"1 ; 1090, 1267, 1430, 1681 (>C=O stretch), 2958, 3059 (O-H stretch); ¹ H NMR (500 MHz, CDCl ₃ ): δ ppm -0.09 (s, 3H), -0.08 (s, 3H) ₃ -0.017 (s, 3H), -0.009 (s, 3H), 0.729 (s, 9H), 0.78 (s, 9H), 1.22 (m, 2H), 1.3 (m, 60H), 1.84 (m, 6H), 2.13 (m, 5H), 2.72 (m, 3H), 2.80 (m, 4H), 3.1-3.3 (m, 4H), 3.0-3.9 (m, 13H), 4.1 l(m, 2H), 4.23 (m, 2H), 4.71 (m, IH), 5.01 (m, IH), 6.01 (m, IH), 7.02 (s, IH), 7.68 (s, IH), 8.01 (m, 6H), 8.17 (m, IH), 10.0 (m, IH): ¹⁹ FNMR (470MHz, CDCl ₃ ): δ ppm -73 (s, 18F).

(6) Preparation of TBDMS-free conjugate

453. To a solution of 4 (80 ing, 0.034 mmol) in DMF (1 ml) was added a solution (0.5 M) of tetrabutylammonium fluoride (1.68 ml, 0.84 mmol) in DMF. After 1 h, the reaction was diluted with EtOAc (25 ml) and poured into a separatory funnel containing saturated NaHCO ₃ (15 ml). The aqueous layer was extracted with EtOAc (5 x 15 ml). The combined organic layers were washed with brine (15 ml), dried over Na ₂ SO ₄ , filtered, and concentrated under vacuum. Flash chromatography with 0-10% MeOH in CH ₂ Cl ₂ yielded TBDMS-free nucleoside (55 mg, 76%). IR (recorded on KBr pellet) v cm ^"1 ; 1040, 1170, 1253, 1525, 1690(XX> stretching), 2970, 3433(O-H stretching); ¹ HNMR (500 MHz, DMSO-D6): δ ppm 0.89 (m, IH), 1.22 (m, 2H), 1.3 (m, 60H), 1.84 (m, 6H), 2.13 (m, 5H), 2.72 (m, 3H), 2.80 (m, 4H), 3.1-3.3 (m, 4H), 3.0- 3.9 (m, 13H), 4.1 l(m, 2H), 4.23 (m, 2H), 4.71 (m, IH), 5.01 (m, IH), 6.01 (m, IH), 7.02 (s, IH), 7.68 (s, IH), 8.01 (m, 6H), 8.17 (m, IH), 10.0 (m, IH): ¹⁹ FNMR (470 MHz, CDCl ₃ ): δ ppm -72 (s, 18F).

(7) Preparation of 5 ^f -0-DimethoxytrityIated 5

454. To a solution of TBDMS-free nucleoside (50 mg, 0.023 mmol) in pyridine (6 ml) was added dimethoxytrityl chloride (10 mg, 0.027 mmol). The reaction was allowed to stir overnight at 25 °C, after which it was quenched with excess methanol. The reaction mixture was diluted with EtOAc (30 ml) and poured into a separatory funnel containing saturated NaHCO ₃ (50 ml). The aqueous layer was extracted with EtOAc (2x15 ml). The combined organic layers were washed with brine (15 ml), dried over Na ₂ SO ₄ , filtered, and concentrated under vacuum. Chromatography (EtOAc) afforded 5 (48 mg, 78%). The material was used in this form during the phosphoramidite preparation (next step). IR (recorded on KBr pellet) v cm ^"1 ; 1050, 1180, 1267, 1505, 1675 (>C=O stretching), 2992 (C-H stretching), 3399 (O-H stretching); ¹ HNMR (500 MHz, CDCl ₃ ): δ ppm 0.89 (m, IH), 1.21 (m, 2H), 1.3 (m, 60H), 1.51 (m, 6H), 2.30 (m, 3H), 2.42 (m, 5H), 2.72 (m, 2H), 2.80 (m, 2H), 3.1-3.3 (m, 6H), 3.3-3.9 (m, 18H), 4.11(m, 2H), 4.23 (m, 2H), 4.61 (m, IH), 4.71 (m, IH) 4.90 (m, IH), 6.01 (m, IH), 6.3 (m, 2H), 6.85 (m, 4H), 7.25 (m, 4H), 7.28 (m, 5H), 8.17 (m, IH), 10.0 (m, IH): ¹⁹ FNMR (470 MHz, CDCl ₃ ): δ ppm

(s, 18F). Compound 5 was treated with -cyanoethyl-N,N,N',N'-tetraisopropyl phosphoramidite in the presence of bis-(diisopropylammonium) tetrazolide to give phosphoramidite 6, which was used without purification for DNA synthesis.

(8) Covalent attachment of neomycin to an oligonucleotide

455. The oligonucleotide synthesis (lμM) was carried out on Expedite Nucleic Acid Synthesis System (8909) using standard phosphoramidite chemistry. The coupling of deoxyuridine-neomycin conjugate 6 (0.2M) to the oligomer was carried out for 30 min. After the oligomer synthesis, the CPG column was dried using argon gas. Then, the conjugate was detached from the solid support using NH ₄ OH and the resulting solution was evaporated. The , lyophilized sample was treated with 1 ml of 1,4-dioxane solution containing 3% CF ₃ CO ₂ H and 1% m-cresol (v/v/v %). After 30 min, the conjugate (Figure 64) was precipitated with 10ml of diethyl ether and the precipitate waa washed with 3x10 ml of diethyl ether. The solution was evaporated and the residue was re-suspended in triethylammonium acetate buffer 1OmM and extracted twice with ether. Ether was. removed and the aqueous layer was dried under vacuum for Ih. The solid was re-dissolved in ImI of water, purified by anion-exchange chromatography, and lyophilized. b) Results

456. The amino groups on rings I, π and TV (Figure 1) of neomycin are necessary in stabilizing and recognizing various nucleic acid forms (aminoglycosides without any of these amines do not stabilize DNA/RNA triplexes and hybrid triplexes as efficiently) (Arya, D. P., et al. Bioorg. Med. Chem. Lett. 2000, 10, 1897-1899; Arya, D. P., et at. J. Am. Chem. Soc. 2001, 123, 11093-11094; Arya, D. P., et al. J. Am. Chem. Soc. 2001, 123, 5385-5395). Therefore, the 5"-OH on ring IU of neomycin was modified to neomycin isothiocyanate 2 as described in our earlier report (Charles, L, et al. Bioorg. Med. Chem. Lett. 2002, 12, 1259-1262). The precursor 1 was prepared from 2'-deoxyuridine by following a literature procedure (Kahl, J. D., et al. J. Am. Chem. Soc. 1999, 121, 597-604). Then, neomycin isothiocyanate 2 was coupled with hexynylamino group of the modified 2'-deoxyuridine 1 to give a conjugate 3 with an isothiourea linkage (Figure 63). The hydroxyl groups on neomycin molecule need to be protected in order to use phosphoramidite chemistry. The hydroxyl groups on a similar kind of aminoglycoside can be protected with acetyl group (Tona, R., et al. J. Org. Lett. 2000, 2, 1693-1696). Deacylation can be carried out at the end of the oligonucleotide-conjugate synthesis with aqueous ammonia treatment, which can also perform the following in a single step: cleavage of the oligonucleotide from the solid support, and β-elimination. Thus, the hydroxyl groups on the neomycin were

protected with trifluoroacetyl group to get acetylated neomycin 4 in 75% yields. Deprotection of t-butyl dimethylsilyl (TBDMS) groups on the sugar with tetrabutylammonium fluoride (TBAF) followed by protection of 5 -hydroxy group with DMTr-Cl in the presence of DMAP in pyridine gave the required TBDMS-free compound 5 in 78% yield. Further reaction of the above compound with 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphoramidite in the presence of bis- (diisopropylammonium) tetrazolide gave the phosphoramidite 6, which decomposed on silica and was thus used without purification (Bannwarth, W. HeIv. Chim. Acta 1988, 71, 1517).

457. After synthesizing the phosphoramidite 6, the 7 mer oligonucleotide (Figure 62) was prepared on a CPG column using standard phosphoramidite synthesis protocols. After introducing three bases, the modified base was coupled with the oligonucleotide on the solid support for 30 min, which was followed by the addition of remaining three bases. Then, the conjugate was detached from the solid support using NH ₄ OH and purified by preparative reverse phase HPLC using a triethylammonium acetate buffer. The dried sample was treated with 1,4-dioxane solution containing 3% CF ₃ CO ₂ H and 1% m-cresol (v/v/v %). After 30 min, the deprotected conjugate was precipitated and washed with excess diethyl ether. The deprotected conjugate was finally purified by preparative anion exchange HPLC using a Tris.HCl buffer (Figure 65). The identity of the conjugate (N-dY) was confirmed by MALDI TOF mass spectrometry (Figure 66).

458. The base composition of the conjugate was determined by complete enzymatic hydrolysis using snake- venom phosphodiesterase followed by alkaline phosphatase and subsequent reversed-phase HPLC chromatography ²⁹ . Thus, the modified and unmodified oligonucleotides (0.2 A ₂₆₀ unit) were subjected to digestion with snake venom phosphodiesterase (10 units/mL) and alkaline phosphatase (100 units/mL) in 50 μL of 50 mM Tris-HCl buffer (pH 7.2) containing 10 mM MgCl ₂ at 37 "C for 24 h. The reaction mixtures were analyzed by reversed phase HPLC (Figure 67).

459. Neomycin-7mer DNA conjugate was generated from a NMR solution structure file (PDB: 124D). The structure was optimized with Maestro program using the AMBER* force field to a gradient of 0.05 kJ/mol A. The all atom AMBER* force field was used as it reproduces x-ray and NMR derived DNA structures. The continuum GB/SA model of water, as implemented in MacroModel, has been used in all calculations. The force field atomic charges were used for triplex. Neomycin was built and optimized in MacroModel as described (Arya, D. P., et al. J. Am. Chem. Soc. 2003, 125, 3733-3744). Then neomycin was conjugated to uridine present in one of the sequence with the same linker used in the present study (Figure 62).

460. Table 5 shows the base sequences used in the present study (X-Indicates the presence of neomycin conjugated to 2'-deoxyuridine at the 5-position).

461. Whether the stabilization shown by neomycin can also be observed with this neomycin-DNA conjugate was then evaluated. Covalent conjugation of a large molecule like neomycin can easily lead to unfavorable base^ackbone contacts, if the molecule is not placed in the major groove with needed flexibility. The thermal denaturation of the rR:dY duplex in the absence/presence of 4 μM of neomycin and presence of various concentration of sodium ion was carried out (Table 6).

462. Table 6. UV melting profiles of the 7 mer hybrid duplex in the presence of various concentration of NaCl. Solution conditions: [DNA]= 4 mM per strand; 10 mM sodium cacodylate buffer, 0.5 mM EDTA, pH 7.0 Samples were heated from 5-90°C at 0.2 deg/min, and were brought back to 20 ⁰ C at a rate of 5 deg/min.

463. In the absence of NaCl, the hybrid duplex (rR:dY) melts at 26.3°C. As the NaCl concentration was increased (0 - 100 mM) the T _n , of the hybrid duplex increased from 26.3°C to 30.0°C. With 60 mM NaCl, the hybrid duplex showed a T _m of 28.1 °C. The presence of 4 μM of neomycin at 60 mM NaCl increases the T _m of the hybrid duplex (rR:dY) by 7.2°C (Figure 68). When the DNA strand (dY) was replaced with the modified oligonucleotide-neomycin conjugate (N-dY), the δT _m of the hybrid duplex (rR:N-dY) was 6.9°C (with 60 mM NaCl) compared to the hybrid duplex (rR:dY) without neomycin. The effect of neomycin on the presence of a mismatch in this duplex (mismatch present on strand-rR ¹ ) was also carried out. The mismatch was chosen on the RNA base which is complementary to the modified base. The presence of a single mismatch on the RNA strand decreases the melting temperature of the hybrid duplex (rR'rdY)

from 35.3°C to 8°C when there is 4 μM of neomycin and 60 mM NaCl present in the solution. The mismatch penalty was similarly observed with the hybrid duplex (rR':N-dY) [with neomycin-oligonucleotide conjugate (N-dY) as one of the DNA strand] showing a T _m of 8°C. This clearly suggests that neomycin's presence does not disrupt the Watson-crick hybridization, and the stabilization seen by neomycin is dependent on the retention of this fidelity leading to the A-form hybrid duplex.

464. The interaction of the aminoglycoside with hybrid nucleic acids can be monitored by CD spectroscopy. Depending upon the nature of the spectrum obtained from the CD scan, one can predict whether the complex exists in A-, B- or canonical forms. There is a preference of aminoglycoside over nucleic acids with A-like conformation (Arya, D. P., et al. J. Am. Chem. Soc. 2001, 123, 5385-5395; Chen, Q., et al. Biochemistry 1997, 36, 11402-11407; Arnott, S., et al. Journal of Molecular Biology 1974, 88, 509-521; Arnott, S., et al. Nucleic Acids Research 1976, 3, 2459-2470). In some cases, transition of B to A conformation was also observed with aminoglycosides, a phenomenon observed with cationic ligands such as spermine and Co(NH ₃ ) ₆ ³⁺ (Robinson, H., et al. Nucleic Acids Research 1996, 24, 676-6S2; Xu, Q., et al. Biophysical Journal 1993, 65, 1039-1049). In a recent report, a complex formed between an 8- basepair hybrid duplex and paramomycin exists in more of A-like canonical conformation (Barbieri, C. M., et al. J. Am. Chem. Soc. 2003, 125, 6469-6477). Previous, competition dialysis results have shown the preference of neomycin conjugates for A-form nucleic acids. The CD spectra of both the hybrid duplexes ((rR ^] :dY and (rR'rN-dY) were analyzed. The spectra showed that the conjugate shifts maximum in the CD spectrum from 275nm to 271nm when compared to non-conjugated hybrid duplex (Figure 69). This shows that the conjugate prefers to be in canonical A-form (Barbieri, C. M., et al. J. Am. Chem. Soc. 2003, 125, 6469-6477; Ivanov, V. L, et al. Biopolymers 1973, 12, 89-110; Gray, D. M., et al. Methods in Enzymology 1992, 211, 389- 406; Gray, D. M., et al. Biopolymers 1975, 14, 487-498) and retains the form exhibited by the unmodified RNA.DNA hybrid duplex.

465. The effect of neomycin binding to the host rR:dY duplex was also studied using ITC experiments. The experiment was conducted at 15°C in sodium cacodylate buffer at pH 6.0. Each injection gave the corresponding heat burst curve and integration of the areas under these curves corresponds to the associated injection heats. These injection heats were subtracted from the dilution heats, which were obtained by two separate blank titrations of the corresponding drug vs buffer and the duplex vs buffer.

466. The thermodynamic properties for the binding of the conjugate N-dY with RNA (rR) were determined with the help of isothermal calorimetry. Monophasic transition was observed when N-dY conjugate was titrated against rR and the injection heat data was fit with a model for single binding site. For comparison, a titration of rR against non-conjugated DNA (dY) was also carried also out and the results are presented in Table 7.

467. Table 7. Binding parameters for the complexation of the rR SS with N-dY SS in cacodylate buffer at pH 7.0 and a temperature of 10 ⁰ C obtained from ITC.

468. The results show that rR single strand prefers to form the duplex approximately two times more with N-dY conjugate more than the noncoηjugated dY single strand. The binding constant for aminoglycoside binding to a hybrid duplex have been previously reported to be in the range of 10 ⁷ M ^"1 (Barbieri, C. M., et al. J. Am. Chem. Soc. 2003, 125, 6469-6477). A two fold change reflects the synergistic effect of DNA and covalently bound neomycin on the duplex structure. The increased stability of the duplex is driven mainly by a larger and more negative enthalpy {δδH _O bs ~ -7.21 kcal/mol (δH _ne omycin conjugated - δH _non conjugated)}. This increase in enthalpy matches the enthalpy of interaction of neomycin binding to the hybrid duplex (Figure 70).

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G. Sequences

SEQ ID NO:l-(-153/-116 upstream of the c-myc gene) GTGGTGGGGTGGTTGGGGTGGGTGGGGTGGGTGGGGT SEQ ID NO:2 (-153/-116 upstream of the c-myc gene) TGGGGTGGGTGGGGTGGGTGGGGTTGGTGGGGTGGTG SEQ ID NO:3 (-142/-115 upstream of the c-myc gene) GGTTGGGGTGGGTGGGGTGGGTGGGGT SEQ ID NO:4 (-142/-115 upstream of the c-myc gene) TGGGGTGGGTGGGGTGGGTGGGGTTGG SEQ ID NO:5 (-61/-16 upstream of the c-myc gene)

TGGTGTGTGGGTTTTGTGGGGGGTGGGGGGGTTTTTTTTGGGTGGG SEQ ID NO:6 (-61/-16 upstream of the c-myc gene)

GGGTGGGTTTTTTTTGGGGGGGTGGGGGGTGTTTTGGGTGTGTGGT SEQ ID NO:7 (-87A58 upstream of the c-myc gene) TTTGGTGTGGGGGTGGGGGTTTTGTTTTTTGT

SEQ ID NO:8 (-87A-58 upstream of the c-myc gene)

TGTTTTTTGTTTTGGGGGTGGGGGTGTGGTTT

SEQ ID NO:9 (-48/-16 upstream of the collagenase gene)

GGTTGGGGTTGGTGTGTTTTTTTTGTGTGGGTG

SEQ ID NO:10 (-48/-16 upstream of the collagenase gene)

GTGGGTGTGTTTTTTTTGTGTGGTTGGGGTTGG

SEQ ID NO:11. (-91/-64 upstream of the collagenase gene)

TTGTGGTTGTTTTTTTGGTTGTGTGTGT

SEQ ID NO:12. (-91/-64 upstream of the collagenase gene)

TGTGTGTGTTGGTTTTTTTGTTGGTGTT

SEQ ID NO:13 (-80/-51 upstream of the HIV-I gene)

TGGGTGGGGTGGGGTGGGGGGGTGTGGGGTGTGGGG

SEQ ID NO:14 (-8O/-51 upstream of the HIV-I gene)

GGGGTGTGGGGTGTGGGGGGGTGGGGTGGGGTGGGT

SEQ ID NO:15 (-16/+13 upstream of the HIV-I gene)

GTTTTTGGGTGTTGTGGGTGTGTGTGGTT

SEQ ID NO:16 (-16/+13 upstream of the HIV-I gene)

TTGGTGTGTGTGGGTGTTGTGGGTTTTTG

SEQ ID NO:17 (-712/-679 upstream of the APP770 gene)

TTTTTGTTTGTTTTTTTTTTGTTTGTTTGTTTT

SEQ ID NO:18 (-712/-679 upstream of the APP770 gene)

TTTTGTTTGTTTGTTTTTTTTTTGTTTGTTTTT

SEQ ID NO:19 (-618/-590 upstream of the APP770 gene)

TGGTGGGGGTTGGTGGTTTGGTTGGTTGT

SEQ ID NO:20 (-618/-59O upstream of the APP770 gene)

TGTTGGTTGGTTTGGTGGTTGGGGGTGGT

SEQ ID NO:21 (-477/-440 upstream of the APP770 gene)

TTGTGTTTGTGTTGGTGTTTGGGGTGGGGGTGGTGTGG

SEQ ID NO:22 (-477/-440 upstream of the APP770 gene)

GGTGTGGTGGGGGTGGGGTTTGTGGTTGTGTTTGTGTT

SEQ ID NO:23 (-434/-407 upstream of the APP770 gene)

GTGTGTTTTTTGGTTTTGGGGTTTTTTT

SEQ ID NO:24 (-434/-407 upstream of the APP770 gene)

TTTTTTTGGGGTTTTGGTTTTTTGTGTG

SEQ ID NO:25 (-286λ252 upstream of the APP770 gene)

GTGTGGTTTGGGTGTTGGTGGTGGGTGGGTGTGGT

SEQ ID NO:26 (-286λ252 upstream of the APP770 gene)

TGGTGTGGGTGGGTGGTGGTTGTGGGTTTGGTGTG

SEQ ID NO:27 (-264/-230 upstream of the APP770 gene)

GGGTGGGTGTGGTGGGGGGTGTGTGTGGGTGGG

SEQ ID NO:28 (-264/-230 upstream of the APP770 gene)

GGGTGGGTGTGTGTGGGGGGTGGTGTGGGTGGG

SEQ ID NO.-29 (-200/-177 upstream of the APP770 gene)

GGGGTGGGGTGGGGGGGGGTGGGG

SEQ ID NO:30 (-200/-177 upstream of the APP770 gene)

GGGGTGGGGGGGGGTGGGGTGGGG

SEQ ID NO:31 (-40/-9 upstream of the APP770 gene)

GTGGGGTGGGTGTGTGGGGGGGGGGGGGGGTG

SEQ ID NO:32 (-40/-9 upstream of the APP770 gene)

GTGGGGGGGGGGGGGGGTGTGTGGGTGGGGTG

SEQ ID NO:33 (-109/-83 upstream of the EGFR gene)

TGGGGGGGTGTGGGGGGGTGGGGGGG

SEQ ID NO:34 (-109/-83 upstream of the EGFR gene)

GGGGGGGTGGGGGGGTGTGGGGGGGT

SEQ ID NO:35 (-307/-281 upstream of the EGFR gene)

TGGGTGGTGGTGGGGGGGTGGGTGGG

SEQ ID NO:36 (-307/-281 upstream of the EGFR gene)

GGGTGGGTGGGGGGGTGGTGGTGGGT

SEQ ID NO:37 (-352/-317 upstream of the EGFR gene)

TTGTGGTGGTGGTGTGGTGGTGGGGTTGGGTGGTGG

SEQ ID NO:38 (-352/-317 upstream of the EGFR gene)

GGTGGTGGGTTGGGGTGGTGGTGTGGTGGTGGTGTT

SEQ ID NO:39 (-363A338 upstream of the EGFR gene)

TTGTGGTGGGTGGTGGTGGGTGGGTGGTGGTGGTGT

SEQ ID NO:40 (-363A338 upstream of the EGFR gene)

TGTGGTGGTGGTGGGTGGGTGGTGGTGGGTGGTGTT

SEQ ID NO:41 (-68A39 upstream of the GSTpi gene)

GTGTGTGGTGTGGGGGGGTGGGGGGGGGGT

SEQ ID NO:42 (-68A39 upstream of the GSTpi gene)

TGGGGGGGGGGTGGGGGGGTGTGGTGTGTG

SEQ ID NO:43 (-227/-204 upstream of the GSTpi gene)

GGGGTGGTGGGTTTGTGGGTTTGG

SEQ ID NO:44 (-227/-204 upstream of the GSTpi gene)

GGTTTGGGTGTTTGGGTGGTGGGG

SEQ ID NO:45 (-499/-410 upstream of the GSTpi gene)

SEQ ID NO:46 (-167/-135 upstream of the HMGCoA Reductase gene)

GGTGTGTGTTGGTGGGGTGGGGGTTGTGGGGGG

SEQ ID NO:47 (-167/-135 upstream of the HMGCoA Reductase gene)

GGGGGGTGTTGGGGGTGGGGTGGTTGTGTGTGG

SEQ ID NO:48 (-134/-104 upstream of the HMGCoA Reductase gene)

GGGTGGGTGGTGTGGGGGGTTGTTTTGGGGT

SEQ ID NO:49 (-134/-104 upstream of the HMGCoA Reductase gene)

TGGGGTTTTGTTGGGGGGTGTGGTGGGTGGG

SEQ ID NO:50 (-41/-6 upstream of the HMGCoA Reductase gene)

TGGGGTTGGGTGGTTGGTTTGTGTTTGGGGGGGGG

SEQ ID NO:51 (-41/-6 upstream of the HMGCoA Reductase gene)

GGGGGGGGGTTTGTGTTTGGTTGGTGGGTTGGGGT

SEQ ID NO:52 (-323/-290 upstream of the NGFR gene)

GGGTTGTGGGTTGGTGGGGGGGTTGGGTGTGTGG

SEQ ID NO:53 (-323/-290 upstream of the NGFR gene)

GGTGTGTGGGTTGGGGGGGTGGTTGGGTGTTGGG

SEQ ID NO:54 (-309/-275 upstream of the NGFR gene)

TGGGGGGGTTGGGTGTGTGGGTGTTTGGGTGTTGG

SEQ ID NO:55 (-309/-275 upstream of the NGFR gene)

GGTTGTGGGTTTGTGGGTGTGTGGGTTGGGGGGGT

SEQ ID NO:56 (-285A248 upstream of the NGFR gene)

TTGGGTGTTGGGTGGGTGTTGGGGTGGGGTGGGGGGT

SEQ ID NO-.57 (-285A248 upstream of the NGFR gene)

TGGGGGGTGGGGTGGGGTTGTGGGTGGGTTGTGGGTT

SEQ ID NO:58 (-243/-216 upstream of the NGFR gene)

GGGTGGGTTTGGGTGTGGTTGGGTGGGG

SEQ ID NO:59 (-243/-216 upstream of the NGFR gene)

GGGGTGGGTTGGTGTGGGTTTGGGTGGG

SEQ ID NO:60 (-60/-26 upstream of the HSV-I polymerase promoter gene)

TTTTTGTGTTGGGGGGTGGGGTGTGGGGGGTGTTT

SEQ ID NO:61 (-60/-26 upstream of the HSV-I polymerase promoter gene)

TTTGTGGGGGGTGTGGGGTGGGGGGTTGTGTTTTT

SEQ ID NO:62 (-82/-118 upstream of the HSV-I polymerase promoter gene)

TTTTTGGGGGGGGGGGGGGGGTGGGTGTTGGGGTGGG

SEQ ID NO:63 (-82/-118 upstream of the HSV-I polymerase promoter gene)

GGGTGGGGTTGTGGGTGGGGGGGGGGGGGGGGTTTTT

SEQ ID NO:64 (+10/+47 / -48/-10 relative to HSV-I origin)

TTTTTTTGTGTGTTGGGGTTGGGTTGGGTGTTTGTGGT

SEQ ID NO-.65 (+10/+47 / -48/-10 relative to HSV-I origin)

TGGTGTTTGTGGGTTGGGTTGGGGTTGTGTGTTTTTTT

SEQ ID NO:66 (+69/-34 relative to HSV-I origin)

TTGGGGGGGGGGGGGGGGGGTTTTTGTTGTGTGTT

SEQ ID NO:67 (+69A34 relative to HSV-I origin)

TTGTGTGTTGTTTTTGGGGGGGGGGGGGGGGGGTT

SEQ ID NO:68 (-912/-886 upstream of the beta globin gene)

GGTTTTGGGGTGGTTGGGGTTGTTTGT

SEQ ID NO:69 (-912/-886 upstream of the beta globin gene)

TGTTTGTTGGGGTTGGTGGGGTTTTGG

SEQ ID NO:70 (-63/-2S upstream of the beta globin gene)

TGGTGGTGGGTGGGGTGGTGGGTGGGGTGGGGTTTTTTG

SEQ ID NO:71 (-63A25 upstream of the beta globin gene)

GTTTTTTGGGGTGGGGTGGGTGGTGGGGTGGGTGGTGGT

SEQ ID NO:72 (-36A9 upstream of the beta globin gene)

TGGGGTGGGGTTTTTTGTGTGGGGTGTG

SEQ ID NO:73 (-36A9 upstream of the beta globin gene)

GTGTGGGGTGTGTTTTTTGGGGTGGGGT

SEQ ID NO:74 (+514/+543 upstream of the beta globin gene)

GGGTTGTTGTTTTGTTTGGGGTTGTTTTGT

SEQ ID NO:75 (+514/+543 upstream of the beta globin gene)

TGTTTTGTTGGGGTTTGTTTTGTTGTTGGG

SEQ ID NO:76 (+693/+719 upstream of the beta globin gene)

TTGTTGGTTTGTTTTTTTTTGTTGTGG

SEQ ID NO-.77 (+693/+719 upstream of the beta globin gene)

GGTGTTGTTTTTTTTTGTTTGGTTGTT

SEQ ID NO:78 (+874/+900 upstream of the beta globin gene)

GTGGGTTGTTTTTTTTGTTTTTTTTTT

SEQ ID NO:79 (+874/+900 upstream of the beta globin gene)

TTTTTTTTTTGTTTTTTTTGTTGGGTG

SEQ ID NO:80 (-162/-141 upstream of the IL-15 gene)

AAGGGAAAGAAAGAAAAAGAA

SEQ ID NO:81 (-162/-141 upstream of the IL-15 gene)

AAGAAAAAGAAAGAAAGGGAA

SEQ ID NO:82 (-1353/-1337 upstream of the pl20 gene)

GAAAAAAGAAGAGAGAA

SEQ ID NO:83 (-1353/-1337 upstream of the pl20 gene)

AAGAGAGAAGAAAAAAG

SEQ ID NO:84 (168/-124 upstream of the human αl(I) collagen gene)

TGGGTTGGGTGGTGGTGGGGGTGTGGTTTGGTTGTGGGTTTTT

SEQ ID NO:85 (-168/-124 upstream of the human αl(I) collagen gene)

TTTTTGGGTGTTGGTTTGGTGTGGGGGTGGTGGTGGGTTGGGT

SEQ ID NO:86 (-294/-264 upstream of the human αl(I) collagen gene)

GGGTTGGGTGTGGTTTGGGGTGGGGTTTGG

SEQ ID NO:87 (-294A264 upstream of the human αl(I) collagen gene)

GGTTTGGGGTGGGGTTTGGTGTGGGTTGGG

SEQ ID NO:88

CGCAAAUUUGCG

SEQ ID NO:89

CGCAAGCUUGCG

SEQ ID NO:90

CGCAAATTTGCG

SEQ ID NO:91

AGGAGAG

SEQ ID NO:92

AGGUGAG

SEQ ID NO:93

AGGAGAG

SEQ ID NO:94

CTCTCCT

SEQ ID NO:95

TCCTCTC

SEQ ID NO:96

HTCTCCCTCTCLysNH2

SEQ ID NO:97

HAGCGTGCGCCATCCCLysNH2

SEQ ID NO:98

HAGATCTTGGAGTGCGLysNH2

SEQ ID NO:99

HTAAACLysNH2

SEQ ID NOrIOO

HCCTAGGAGGAATLysNH2

SEQ ID NOrIOl

HCCGGCNH2