Login| Sign Up| Help| Contact|

Patent Searching and Data


Title:
MODIFIED NUCLEOMONOMERS AND METHODS OF USE THEREOF
Document Type and Number:
WIPO Patent Application WO/2000/011013
Kind Code:
A1
Abstract:
Modified 5'-unsubstituted 2' deoxyuridine residues appended with 3-aminopropyl,3-hydroxylpropyl and 3-aminopropyn-1-yl- sidechains have been used to study the effects of localized charges on duplex DNA oligomers. It has been shown that the 3-aminopropyl substitutions induce a proximately 8° bend in the duplex and it was assumed that the tethered amine formed a salt bridge with the 5' phosphate. However, the characterization of different appendages indicate that only the 3-aminopropyn-1-yl-sidechain can form a salt bridge.

Inventors:
GOLD BARRY (US)
Application Number:
PCT/US1999/019029
Publication Date:
March 02, 2000
Filing Date:
August 20, 1999
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
UNIV NEBRASKA (US)
GOLD BARRY (US)
International Classes:
C07H15/18; C07H19/10; C12N15/113; A61K38/00; (IPC1-7): C07H21/04; A61K48/00; C07H21/02
Other References:
ROBINS ET AL: "Nucleic Acid Related Compounds. 39. Efficient Conversion of 5-Iodo to 5-Alkynyl and Derived 5-Substituted Uracil Bases and Nucleosides", J. ORG. CHEM., vol. 48, no. 11, January 1983 (1983-01-01), pages 1854 - 1862, XP002069924
SZABOLCS ET AL: "Unnatural Nucleosides and Nucleotides, III, Preparation of 2-(14)C and 4-(14)C Labelled 5-Alkyluracils and 5-Alkyl-2'-Deoxyuridines", JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, vol. 14, no. 5, January 1978 (1978-01-01), pages 713 - 726, XP002922335
Attorney, Agent or Firm:
Rigaut, Kathleen D. (Dorfman Herrell and Skillman Suite 720 1601 Market Street Philadelphia, PA, US)
Download PDF:
Claims:
What is claimed is:
1. A modified nucleomonomer having a formula selected from the group consisting of formulas (I) and (II), wherein X is a divalent linking moiety selected from the group consisting ofC=C (CH2) m,CH=CH (CHZ) n, or (CH2) p ; m and n are integers from 1 to 2 and p is an integer from 3 to 4; R is a substituent group selected from those consisting of OH, NH2, SH, alkoxy (ClC4) and alkythio (C1C4) and pharmaceutically acceptable salts thereof.
2. A nucleomonomer as claimed in claim 1, wherein X isC=C (CHZ) m, m is 1 and R is OH.
3. An oligomer comprising at least one nucleomonomer as claimed in claim 1.
4. An oligomer as claimed in claim 3, said oligomer being between 2 and 50 nucleomonomers in length.
5. An oligomer as claimed in claim 3, said oligomer having a conventional phosphodiester linkage.
6. An oligomer as claimed in claim 3, said oligomer having a substitute linkage selected from the group consisting of phosphorothioate, methylphosphonate and thionomethylphosphonate linkages.
7. A pharmaceutically acceptable salt of an oligomer as claimed in claim 3.
8. A composition comprising an oligomer as claimed in claim 3 and a biologically compatible carrier medium.
9. A composition as claimed in claim 8, wherein said composition further comprises an agent for improving membrane permeability of said oligomer.
10. A composition as claimed in claim 8, wherein said biologically compatible medium contains at least one targeting agent for effecting delivery of said oligomer to said cells.
11. A composition as claimed in claim 10, wherein said at least one targeting agent comprises a liposome having binding affinity for said cells, and said oligomer is encapsulated within said liposome.
12. A method for inhibiting expression of a nucleic acid molecule comprising: a) providing an oligomer of complementary sequence substituted with a nucleomonomer as claimed in claim 1; and b) administering said substituted oligomer to a cell containing said nucleic acid molecule in an effective amount to inhibit expression of said nucleic acid.
13. A method as claimed in claim 12, wherein said nucleic acid is DNA.
14. A method as claimed in claim 12, wherein said nucleic acid is RNA.
15. A method as claimed in claim 12, wherein said cell is selected from the group consisting of bacterial cells, fungal cells, yeast cells, mammalian cells, cancer cells, and virally infected cells.
Description:
MODIFIED NUCLEOMONOMERS AND METHODS OF USE THEREOF This application claims priority under 35 U. S. C.

§119 (e) from U. S. Provisional Application 60/097,712 filed August 22,1998, the entire disclosure of which is incorporated by reference herein.

FIELD OF THE INVENTION This invention relates to the fields of gene regulaticn and antisense technology. More specifically, modified nucleomonomers are provided which are incorporated into antisense oligomers. Such oligomers demonstrate increased duplex DNA stability when hybridizing to target nucleic acid sequences. Methods employing the modified nucleomonomers of the invention are also provided.

BACKGROUND OF THE INVENTION Several publications are referenced in this application by author name, year and journal of publication in parentheses in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications is incorporated by reference herein.

Antisense oligodeoxynucleotides (ODNs) are capable of blocking expression of target genes in cells.

Antisense technologies offer new therapies for the treatment of human diseases. Gene expression is inhibited ODNs bind to a complementary messenger RNA sequence and prevents its translation. Antisense effects have been reported using ODNs in mammalian cells in tissue culture and in in vivo studies (Weiss, B., editor, =mtisense Oligodeoxynucleotides and Antisense

RNA novel pharmacological and therapeautic agents, CRC Press, Boca Raton, Florida 1997). The efficacy of antisense techniques is currently being investigated in several human diseases including hypertension, Burkitt's lymphoma, and infection by human immunodeficiency virus (Weiss et al., supra).

There are, however, numerous reports of certain unpredictable biological effects associated with ODN treatment that cannot be attributed to the anticipated antisense action alone. In Xenopus, for example, antisense ODNs cause cleavage of imperfectly matched target sites (Woolf, T. M.; Melton, D. A.; Jennings, C.

G. Proc. natn. Acad. Sci. U. S. A. 1992,89,7305-7309).

Sequence-specific and non-specific binding of ODNs to small molecules and proteins has also been reported (Ellington, A. D.; Szostak, J. W. Nature 1990,818-822; Yakubov, L. et al. J. Biol. Chem. 1993,268,18818- 18823). Phosphorothioate ODNs can nonspecifically activate the SP1 transcription factor of a cell (Perez, J. R. et al. Proc. Natl. Acad. Sci. 1994,91,5957- 5961). ODNs can inhibit viral infection by non- antisense methods that may include interference with absorption, penetration or uncoating (Azad, R. F.; Driver, V. B.; Tanaka, K.; Crooke, R. M.; Anderson, K.

P. Antimicrob. Agents Chemother. 1993 37,1945-1954).

Finally, ODNs can affect cell proliferation and differentiation (Kamano, H. et al. Biochem. Int. 1992, 26,537-543). ODNs can be degraded both intra-and extracellularly (Agrawal, S.; Temsamani, J.; Tang, J. Y.

Proc. Natl. Acad. Sci. U. S. A. 1991 88,7595-7599), and these breakdown products may, in part, be responsible for the observed non-antisense effects, particularly when the ODN is used at high concentrations. In order

to ameliorate these non-antisense effects, the present inventors have appreciated a need for ODN's which are physiologically stable, non-toxic, able to penetrate cells, and maintain stringent base-pairing fidelity for unique DNA sequences (Wagner, R. W. Nature 1994,372, 333-335).

SUMMARY OF THE INVENTION In accordance with one aspect, the present invention provides modified nucleomonomers and compositions containing the same for inhibiting or regulating expression of target genes and nucleic acids.

The modified nucleomonomers of the invention are selected from those having the structural formulas (I) and (II) shown below:

wherein X is a divalent linking moiety selected from the group consisting of-C-C-(CH2) CH=CH-(CH2) n-, or -(CH2) p-,(CH2) p-, m and n being integers from 1 to 2 and p being an integer from 3-4 and R is a substituent group selected from those consisting of OH, NH2, SH, alkoxy (C1-C4) and alkythio (C1-C4) and pharmaceutically acceptable salts thereof. Preferably, in the modified nucleomonomers of formulas (I) and (II), m and n are 1, p is 3 and R is OH. Most preferred is a modified nucleomonomer wherein X is-C=C- (CH2) m-and m is 1.

In yet another aspect of the invention, the novel nucleomonomers of the invention are incorporated into oligomer analogues. Such oligomers have the ability to form DNA triplexes and regulate gene expression. Any target sequence may be chosen for regulation provided that the sequence incorporates one of the base analogs of the invention. Suitable sequences for targeting include those from pathogenic bacteria, fungi and viruses, oncogenes, growth hormones, and enzymes.

The oligomers of the invention may be incorporated into pharmaceutically acceptable carriers. Such pharmaceutical compositions are useful in the treatment of disease. The oligomers of the invention may also be used in diagnostic applications to detect target sequences in biological samples.

The following definitions are provided to facilitate understanding of the present invention: Nucleomonomer. As used herein, the term "nucleomonomer"means a moiety comprising (1) a base covalently linked to (2) a second moiety. Nucleomonomers include conventional and chemically modified nucleosides and nucleotides. Nucleomonomers can be linked to form

oligomers that bind to target or complementary base sequences in nucleic acids in a sequence specific manner. A"second moiety"as used herein includes a sugar moiety, such as deoxyribose or ribose.

Base."Base"as used herein includes those moieties which contain not only the known purine and pyrimidine heterocycles but also the modified pyrimidines of the invention. Purines include adenine, guanine and xanthine. Pyrimidines include uracil and cytosine and their analogs Nucleoside. As used herein,"nucleoside"means a base covalently attached to a sugar or sugar analog.

Nucleotide. As used herein,"nucleotide"means nucleoside having a phosphate group or phosphate analog.

Linkage. As used herein,"linkage"means a phosphodiester moiety (--O--P (O) (O)--O--) that covalently couples adjacent nucleomonomers.

Substitute Linkages. As used herein,"substitute linkage"means any analog of the native phosphodiester group that covalently couples adjacent nucleomonomers.

Substitute linkages include phosphodiester analogs, e. g. such as phosphorothioate and methylphosphonate, and nonphosphorus containing linkages, e. g. such as acetals and amides.

Oligomers. Oligomers are defined herein as two or more nucleomonomers covalently coupled to each other by a linkage or substitute linkage moiety. Thus, an

oligomer can have as few as two nucleomonomers (a dimer). Oligomers can be binding competent and, thus, can base pair with cognate single-stranded or double-stranded nucleic acid sequences. Oligomers (e. g. dimers-hexamers) are also useful as building blocks for longer oligomers as described herein. As used herein "oligomer"includes oligonucleotides, oligonucleosides, polydeoxyribo-nucleotides (containing 2'-deoxy-D-ribose or modified forms thereof), i. e., DNA, polyribonucleotides (containing D-ribose or modified forms thereof), or RNA, and any other type of polynucleotide which is an N-glycoside or C-glycoside of a purine or pyrimidine base, or modified purine or pyrimidine base. Oligomer as used herein is also intended to include compounds where adjacent nucleomonomers are linked via amide linkages as previously mentioned (Nielsen, P. E., et al, Science (1991) 254: 1497-1500). The enhanced competence of binding by oligomers containing the modified nucleomonomers of the present invention is believed to be primarily a function of the base alone. Because of this, elements ordinarily found in oligomers, such as the furanose ring and/or the phosphodiester linkage can be replaced with any suitable functionally equivalent element."Oligomer"is thus intended to include any structure that serves as a scaffold or support for the bases wherein the scaffold permits binding to target nucleic acids in a sequence-dependent manner.

Oligomers that are currently known can be defined into four groups that can be characterized as having (i) phosphodiester and phosphodiester analog (phosphorothioate, methylphosphonate, etc) linkages, (ii) substitute linkages that contain a non-phosphorous

isostere (formacetal, riboacetal, carbamate, etc), (iii) morpholino residues, carbocyclic residues or other furanose sugars, such as arabinose, or a hexose in place of ribose or deoxyribose and (iv) nucleomonomers linked via amide bonds or acyclic nucleomonomers linked via any suitable substitute linkage.

The oligomers of the invention can be formed using the modified nucleomonomers of the invention or conventional nucleomonomers and synthesized using standard solid phase (or solution phase) oligomer synthesis techniques, which are now commercially available. In general, the oligomers of the invention can be synthesized by a method comprising the steps of: synthesizing a nucleomonomer or oligomer having a protecting group and a base and a coupling group capable of coupling to a nucleomonomer or oligomer; coupling the nucleomonomer or oligomer to an acceptor nucleomonomer or an acceptor oligomer; removing the protecting group; and repeating the cycle as needed until the desired oligomer is synthesized.

The oligomers of the present invention can be of any length including those of greater than 40,50 or 100 nucleomonomers. In general, preferred oligomers contain 2-30 nucleomonomers. Lengths of greater than or equal to about 8 to 20 nucleomonomers are useful for therapeutic or diagnostic applications. Short oligomers containing 2,3,4 or 5 nucleomonomers are specifically included in the present invention and are useful as starting materials for longer oligomers.

Oligomers having a randomized sequence and containing about 6 or 7 nucleomonomers are useful as primers for cloning or amplification protocols that use random sequence primers, provided that the oligomer

contains residues that can serve as a primer for polymerases or reverse transcriptases.

Oligomers can contain conventional phosphodiester linkages or can contain substitute linkages such as phosphoramidate linkages. These substitute linkages include, but are not limited to, those wherein a moiety of the formula--O--P (O) (S)--O-- ("phosphorothioate"), --O--P (S) (S)--O-- ("phosphorodithioate"), --O--P (O) (NR| 2)--X----O--P (O) (R')--O-- --O--P (S) (R')--O-- ("thionoalkylphosphonate"), --P (O) (OR")--X--,--O--C (O)--X--, or --O--C (O) (NR'2)--X--, wherein R'is H or alkyl (1-12C) and R"is alkyl (1-9C) and the linkage is joined to adjacent nucleomonomers through an--O--or--S--bonded to a carbon of the nucleomonomer. Particularly, preferred substitute linkages for use in the oligomers of the present invention include phosphodiester, phosphorothioate, methylphosphonate and thionomethylphosphonate linkages. Phosphorothioate and methylphosphonate linkages confer added stability to the oligomer in physiological environments. While not all such linkages in the same oligomer need be identical, particularly preferred oligomers of the invention contain uniformly phosphorothioate linkages or uniformly methylphosphonate linkages.

Blocking Groups. As used herein,"blocking group" refers to a substituent other than H that is conventionally attached to oligomers or nucleomonomers, either as a protecting group, a coupling group for synthesis, P03-2, or other conventional conjugate such as a solid support. As used herein,"blocking group"is not intended to be construed solely as a protecting group,

according to common terminology, but also includes, for example, coupling groups such as a hydrogen phosphonate or a phosphoramidite.

Protecting group."Protecting group"as used herein means any group capable of preventing the O-atom or N-atom to which it is attached from participating in a reaction or bonding. Such protecting groups for O-and N-atoms in nucleomonomers are described and methods for their introduction are conventionally known in the art.

Protecting groups also prevent reactions and bonding at carboxylic acids, thiols and the like.

Coupling group."Coupling group"as used herein means any group suitable for generating a linkage or substitute linkage between nucleomonomers such as a hydrogen phosphonate and a phosphoramidite.

Conjugate."Conjugate"as used herein means any group attached to the oligomer at a terminal end or within the oligomer itself. Conjugates include solid supports, such as silica gel, controlled pore glass and polystyrene; labels, such as fluorescent, chemiluminescent, radioactive, enzymatic moieties and reporter groups; oligomer transport agents, such as polycations, serum proteins and glycoproteins, polymers and the like.

Pi bond."Pi bond"as used herein means an unsaturated covalent bond such as a double or triple bond. Both atoms can be carbon or one can be carbon and the other nitrogen, for example, phenyl, propynyl, cyano and the like.

Pharmaceutically acceptable salts. Such"salts" are preferably metal or ammonium salts of the oligomers of the invention and include alkali or alkaline earth metal salts, e. g., the sodium, potassium, magnesium or calcium salt; or advantageously easily crystallizing ammonium salts derived from ammonia or organic amines, such as mono-, di-or tri-lower (alkyl, cycloalkyl or hydroxyalkyl)-amides, lower alkylenediamines or lower (hydroxyalkyl or arylalkyl)-alkylammonium bases, e. g. ethylamine, diethylamine, triethylamine, dicyclohexylamine, triethanolamine, ethylenediamine, tris- (hydroxymethyl)-aminomethane or benzyl-trimethylammonium hydroxide. The oligomers of the invention form acid addition salts, which are preferably therapeutically acceptable inorganic or organic acids, such as strong mineral acids, for example hydrohalic, e. g., hydrochloric or hydrobromic acid; sulfuric, phosphoric; aliphatic or aromatic carboxylic or sulfonic acids, e. g., formic, acetic, propionic, succinic, glycollic, lactic, malic, tartaric, gluconic, citric, ascorbic, maleic, fumaric, hydroxymaleic, pyruvic, phenylacetic, benzoic, 4-aminobenzoic, anthranilic, 4-hydroxybenzoic, salicylic, 4-amino salicylic, methanesulfonic, ethanesulfonic, hydroxy ethanesulfonic, benzenesulfonic, sulfanilic or cyclohexylsulfamic acid and the like.

A"positive modification"is any modification of the nucleomonomer of formula (1) or (2) above which results in increased binding affinity.

A"negative modification"is any nucleomonomer

modification of an oligomer comprising a base of formula (1) or (2) which results in a decrease in binding affinity or use of a substitute linkage which may result in a decrease in binding affinity.

Transfection."Transfection"as used herein refers to any method that is suitable for enhanced delivery of oligomers into cells.

Subject."Subject"as used herein means an animal, including a mammal, particularly a human.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the synthesis of modified deoxyuridine and deoxycytosine phosphoramidites.

Figure 2 illustrates the structure of the universal support utilized to synthesize the oligomers of the invention.

Figure 3 shows a minimized relaxed stereo-view structures of ODN with 3-aminopropyl (Y= 5P-NH2-dU) group pointing toward the 3'direction, and 3-aminopropyn-1-yl (X= 5P=NH2-dU) group pointing toward the phospate on the 5'residue making a salt bridge. The complementary strand of the duplex has been removed for viewing purposes.

DETAILED DESCRIPTION OF THE INVENTION The development of antisense therapies to inhibit the expression of specific genes requires the generation of oligonucleotides (ODNs) that are physiologically stable, nontoxic, and able to penetrate into cells,

while maintaining stringent base pairing fidelity for target DNA sequences (Wagner et al., supra).

Modifications to the bases, deoxyribose ring, and phosphate backbone have been generated in order to satisfy some of these criteria (Milligan, J. F.; Matteucci, M. D.; Martin, J. C. J. Med. Chem. 1993,36, 1923-1937). Among the potential structural changes, alterations of phosphate backbone have received most attention and have had the greatest impact on antisense technology. In fact, backbones made up of phosphorothioates have been tested in vivo as antisense reagents because they are DNase resistant and the resulting RNA-DNA complex is a substrate for RNase H (Fisher, T. L.; Terhorst, T.; Cao, X.; Wagner, R. W.

Nucl. Acids Res. 1993,3857-3865). However, phosphorothioate-based ODNs are complex diastereomeric mixtures, cause toxicity due to mechanisms which are poorly understood, and most importantly, form base pairs with reduced stability (Kibler-Herzog, L.; Zon, G.; Uzanski, B.; Whittier, G.; Wilson, W. D. Nucl. Acids Res. 1991,19,2979-2986).

In accordance with the present invention, modified deoxynucleosides are provided. Representative structures are set forth in formulas (I) and (II) above. Such modified deoxynucleotides may be conveniently synthesized from known starting materials according to the synthetic scheme depicted in Figure 1.

Briefly, the syntheses of 5P=NH2-dU and 5P=OH-dU were accomplished by coupling 5-iodo-2'-deoxyuridine with 1- aminoprop-2-yne (protected as the N-phthalimide) or 1- hydroxyprop-2-yne (protected as the benzoyl ester) in the presence of (PPh),, Pd (o) as previously described (Heystek, L. E., Zhou, H.-q., Dande, P. and Gold, B.

(1998) J. Amer. Chem. Soc. 120,12165-12166; Gibson, K.

J. and Benkovic, S. J. (1987) Nucleic Acids Res. 15, 6455-6467). The deoxynucleosides are converted into the 5'-O-dimethoxytrityl-3'-O-phosphoramidites by standard procedures (Beaucage. S. L. Methods in Molecular Biology, Protocols for oligonucleotides and analogues.

Synthesis and properties. Vol. 20 Agrawal, S., Ed.; Humana, Totowa, NJ, 1993, pp 33-61). The structures were confirmed by'H-NMR and mass spectrometry. The syntheses of the 5P-NH2-dU and 5P-OH-dU analogues were performed using a similar coupling reaction followed by hydrogenation using Pd or Raney-Ni catalysts (Gibson et al., supra). The dC analogues, e. g., 5P--NH2-dC (Figure 1), are prepared using the same procedures except the starting material is 5-iodo-dC and the N'-amino group is protected as the N-benzoyl derivative (Hashimoto, H., Nelson, M. G. and Switzer, C. (1993) J. Org. Chem. 58,4194- 4195). All phosphoramidites were incorporated into oligomers using standard chemistry on an ABI synthesizer, purified by reverse phase HPLC, and shown to be homogeneous by polyacrylamide gel electrophoresis.

To date, the ODNs with 3-aminopropyn-1-yl, 3-aminopropyl and 3-hydroxypropyl sidechains have been characterized by sequential hydrolysis and HPLC analysis (Heystek et al., supra).

In vitro studies have been performed which demonstrate the superior DNA binding ability of the modified oligomers of the invention. The modifications set forth herein dramatically increase the melting point (Tm) thereby mediating a powerful stabilizing effect on triplex formation.

In a preferred embodiment, the compounds of the invention have the following formulae:

wherein X is a divalent linking moiety selected from the group consisting of-C-C-(CH2) m-,-CH=CH-(CH2) n~ or - (CH2) p-, m and n being integers from 1 to 2 and p being an integer from 3-4 and R is a substituent group selected from those consisting of OH, NH2, SH, alkoxy (Cl-C4) and alkythio (C1-C4) and pharmaceutically acceptable salts thereof. Preferably, in the modified

nucleomonomers of formulas (I) and (II), m and n are 1, p is 3 and R is OH. Most preferred is a modified nucleomonomer wherein X is _CEC- (CH2) m-and m is 1.

As the oligomers of the invention demonstrate significant single-stranded or double-stranded target nucleic acid binding activity to form duplexes, triplexes or other forms of stable association, these oligomers are useful in diagnosis and therapy of diseases that are associated with expression of one or more genes, which includes many diverse pathological conditions, as noted below. Therapeutic applications employing the oligomers to specifically inhibit the expression of genes (or inhibit translation of RNA sequences encoded by those genes) that are associated with either the establishment or the maintenance of a pathological condition are also contemplated to be within the scope of the present invention. Exemplary genes or encoded RNAs that can be targeted include those that encode enzymes, hormones, serum proteins, adhesion molecules, receptor molecules, cytokines, oncogenes, growth factors, and interleukins. Target genes or RNAs can be associated with any pathological condition such as inflammatory conditions, cardiovascular disorders, immune reactions, cancer, viral infections, bacterial infections and the like.

Oligomers of the present invention are suitable for both in vivo and ex vivo therapeutic applications.

Indications for ex vivo uses include treatment of cells such as bone marrow or peripheral blood in conditions such as leukemia or viral infection. Genes that can serve as targets for cancer treatments include oncogenes, such as ras, k-ras, bcl-2, c-myb, bcr, c-myc, c-abl or overexpressed sequences such as mdm2,

oncostatin M, IL-6 (Kaposi's sarcoma), HER-2 and translocations such as bcr/abl, or RNAs encoded by such genes. Viral gene sequences, such as polymerase or reverse transcriptase genes of CMV, HSV-1, HSV-2, HTLV-1, HIV-1, HIV-2, HBV, HPV, VZV, influenza virus, rhinovirus and the like or RNAs encoded by these are also suitable targets. Application of specifically binding oligomers can be used in conjunction with other therapeutic treatments. Other therapeutic indications for oligomers of the invention include (1) modulation of inflammatory responses by modulating expression of genes such as IL-1 receptor, IL-1, ICAM-1 or E-Selectin that play a role in mediating inflammation and (2) modulation of cellular proliferation in conditions such as arterial occlusion (restenosis) after angioplasty by modulating the expression of (a) growth or mitogenic factors such as non-muscle myosin, myc, fos, PCNA, PDGF or FGF or their receptors, or (b) cell proliferation factors such as c-myb. Other suitable extracellular proliferation factors such as TGFa, IL-6, yINF, protein kinase C may be targeted for treatment of psoriasis or other conditions. In addition, EGF receptor, TGFa or MHC alleles may be targeted in autoimmune diseases.

Delivery of oligomers of the invention into cells can be enhanced by any suitable method including calcium phosphate, DMSO, glycerol or dextran transfection, electroporation or by the use of cationic anionic and/or neutral lipid compositions or liposomes by methods described, for example, in International Publication Nos. WO 90/14074, WO 91/16024, WO 91/17424, and U. S.

Pat. No. 4,897,355, the disclosure of which are incorporated by reference herein. The oligomers can be introduced into cells by complexation with cationic

lipids such as DOTMA (which may or may not form liposomes), which complex is then contacted with the cells. Suitable cationic lipids include but are not limited to N- (2,3-di (9- (Z)-octadecenyloxyl)) -prop-1-yl-N, N, N-trimethylammonium (DOTMA) and its salts, 1-O-oleyl-2-0-oleyl-3- dimethylaminopropyl-ß-hydroxyethylammonium and its salts and 1,2-bis (oleyloxy)-3- (trimethylammonio) propane and its salts.

Enhanced delivery of the oligomers of the invention can also be mediated by the use of (i) viruses such as Sendai virus (Bartzatt, R., Biotechnol Appl Biochem (1989) 11: 133-135) or adenovirus (Wagner, E., et al, Proc Natl Acad Sci (1992) 89: 6099-6013; (ii) polyamine or polycation conjugates using compounds such as polylysine, protamine or N1, N12-bis (ethyl) spermine (Wagner, E., et al, Proc Natl Acad Sci (1991) 88: 4255-4259; Zenke, M., et al, Proc Natl Acad Sci (1990) 87: 3655-3659; Chank, B. K., et al, Biochem Biophys Res Commun (1988) 157: 264-270; U. S. Pat. No.

5,138,045); (iii) lipopolyamine complexes using compounds such as lipospermine (Behr, J.-P., et al, Proc Natl Acad Sci (1989) 86: 6982-6986; Loeffler, J. P., et al J Neurochem (1990) 54: 1812-1815); (iv) anionic, neutral or pH sensitive lipids using compounds including anionic phospholipids such as phosphatidyl glycerol, cardiolipin, phosphatidic acid or phosphatidylethanolamine (Lee, K.-D., et al, Biochim Biophys ACTA (1992) 1103: 185-197; Cheddar, G., et al, Arch Biochem Biophys (1992) 294: 188-192; Yoshimura, T., et al, Biochem Int (1990) 20: 697-706); (v) conjugates with compounds such as transferrin or biotin or (vi) conjugates with compounds such as serum proteins

(including albumin or antibodies), glycoproteins or polymers (including polyethylene glycol) that enhance pharmacokinetic properties of oligomers in a subject.

Any reagent such as a lipid or any agent such as a virus that can be used in transfection protocols is collectively referred to herein as a"permeation enhancing agent". Delivery of the oligomers into cells can be via cotransfection with other nucleic acids such as (i) expressible DNA fragments encoding a protein (s) or a protein fragment or (ii) translatable RNAs that encode a protein (s) or a protein fragment.

The oligomers can thus be incorporated into any suitable formulation that enhances delivery of the oligomers into cells. Suitable pharmaceutical formulations also include those commonly used in applications where compounds are delivered into cells or tissues by topical administration. Compounds such as polyethylene glycol, propylene glycol, azone, nonoxonyl-9, oleic acid, DMSO, polyamines or lipopolyamines can be used in topical preparations that contain the oligomers.

Thus, the modified nucleomonomers described herein, may be used effectively to inhibit expression of a selected protein or proteins in a subject or in cells, wherein the proteins are encoded by DNA sequences and the proteins are translated from RNA sequences by introducing an oligomer of the invention into the cells; and permitting the oligomer to form a triplex with the DNA or RNA or a duplex with the DNA or RNA whereby expression of the protein or proteins is inhibited. The method is suitable for modulating gene expression in both procaryotic and eucaryotic cells such as bacterial, parasite, yeast and mammalian cells.

The following methods and experimental designs are provided to facilitate the practice of the present invention.

Oligomer design. A series of ODNs are listed below which may be used in the practice of the present invention (Table A). Duplex ODN-A has a d (A) 15: d (T) l5 duplex target for the third strand and the other (ODN-B) contains several G: C base pairs. The synthesis of the phosphoramidites has already been achieved for the dU analogues using the scheme shown in Figure 1. The synthesis of ODNs in which the 3'-residue is modified (see Table A) requires a non-standard approach since the 3'-residue of the ODN is normally directly attached to the solid support. To prepare ODNs in which the 3'- residue has a sidechain modification, the solid support that is used has an attached abasic site with a ribose sugar ring (Figure 2). The ODN synthesis is performed as usual, which affords an ODN with an extra ribose sugar residue on the 3'-terminus. The ribose sugar is removed during the deprotection step by treatment with 0.5 M NaOH in MeOH/H2O (1: 1) for 1 h at R. T as recommended by Glenn Research (Sterling, VA). This approach gives excellent yields using the 5P=-NH2-dU modified DNA. The third strands listed in Table A, which contain modified residues at internal and/or external sites will facilitate the evaluation of the effect that one of more of the modified residues have on the kinetics of triplex formation, triplex stability and Hoogsteen base pairing fidelity.

Table A.

Structure of duplex targets and ODNs'

structure of duplex-A and third structure of duplex-B and third strand strand duplex-A 5'-duplex-B 5'-TGAGAAAAAGAGAGAGAAACCAA TGAGAAAAAAAAAAAAAAACCAA 3'-ACTCTTTTTCTCTCTCTTTGGTT 3'-ACTCTTTTTTTTTTTTTTTGGTT ODN-q 5'-TTTTTCTCTCTCTTT ODN-a 5'-TTTTTTTTTTTTTTT ODN-r 5'-TTTTTXTCTCTCTTT ODN-b 5'-TTTTTTTXTTTTTTT ODN-s 5'-TTTTTXTXTCTCTTT ODN-c 5'-TTTTTXTTTXTTTTT ODN-t 5'-TTTTXXXXXCTCTTT ODN-d S'-XTTTTXTTTXTTTTX ODN-u 5'-XTTTTXTXTTT=TTX ODN-e 5'-XTTTTTTTTTTTTTX ODN-v 5'-XXXXXXXXXXXXXXX ODN-f 5'-XXXXXXXXXXXXXXX ODN-w 5'-TTTTTWTWTCTCTTT ODN-g 5'-TTTTTTTWTTTTTTT ODN-x 5'-WWWWWWWWWWWWWWW ODN-h 5'-TTTTTWTTTWTTTTT ODN-y 5'-TTTTTPTPTCTCTTT ODN-i 5'-WTTTTWTTTWTTTTW ODN-z 5'-PPPPPPPPPPPPPPP ODN-j 5'-WTTTTTTTTTTTTTW ODN-aa 5'-TTTTTYTYTCTCTTT ODN-k 5'-WWWWWWWWWWWWWWW ODN-bb 5'-YYYYYYYYYYYYYYY ODN-1 5'-TTTTTTTPTTTTTTT ODN-m 5'-TTTTTPTTTPTTTTT ODN-n 5'-PTTTTPTTTPTTTTP ODN-o 5'-PTTTTTTTTTTTTTP QDN-p5'-PPPPPPPPPPPPPPP ¹ X = 5P=NH2-dU, 5-(3-aminopropyn-1-yl)dU ; W = 5P OH-dU, 5- (3- . E = 5P=H-dU, 5- (I-propynyl)dU; Y = 5P-NH2-dU, 5- (3-aminopropyl) dU; X = 5P=NH2-dC; 5- (3-aminopropyn-1-yl) dC; W = 5P=OH-dC, 5- (3-hydroxypropyn-1-yl) dC; Y = 5P-NH2-dC, 5- (3- aminopropyl) dC; P = 5P=H-dC, 5-(1-propynyl)dC.

TM studies. The TMstudies can be done as described in Example 1. See legend of Table 2. An alternative approach using a more"physiological"buffer is also contemplated to be within the scope of the present invention. This buffer consists of 25 mM sodium phosphate (pH 7.0) containing 70 mM KC1,2 mM MgCl2, and

400 HM spermine. The TM results are readily converted into binding constants, and thermodynamic properties.

To measure the rate of triplex formation, the length of the incubation of the third strand with the duplex at 4 °C will be varied: 1,3,6,9,12 and 24 h.

The LAbsorbance at 260 and 280 nm as a function of temperature will be used to determine triplex concentration. For the duplex ODNs that contain G: C pairs (Table A, right panel), the TM values will also be determined as a function of pH (5.0,5.5,6.0,6.5,7.0 and 7.5). Normally, unmodified ODN with C residues require acidic pH to form an N3-protonated-dC: the protonated C binds to G via Hoogsteen base pairing.

These pH dependency studies will be done with a 24 h incubation period to allow the triplex to form.

In order to assess the TM results with the G: C targets, the pKa of 5PENH2-dC will be determined. The protonation requirement for dC in the third strand in general limits triplex stability at physiological pH.

The 5P--NH2-dC modifications should stabilize triplex formation. Experiments will also be conducted to determine whether the stabilization is due to protonation and/or the cationic appendage. This will be done by titrating the free 5P NH2-dC with HCl while monitoring the UV: the for dC shifts 10 nm upon protonation (Singer, B. and Grunberger, D. (1983) Molecular Biology of Mutagens and Carcinogens, Plenum Press, N. Y. p 305). 5-Methyl-dC has a pKa of 4.4, while 5P=H-dU has a pKa of 3.5 (Froehler, B. C., Wadwani, S., Terhorst, T. J. and Gerrard, S. R. (1992) Tetrahedron Lett. 33,5307-5310).

Gel Shift assay. The duplex substrates can be shortened

from the 33mers described in Example I to 23mers with shorter ends, and the use of the 32P-labeled ODN should permit resolution of the triplex band more clearly. In the experiments described in Example I below, the duplex was end-labeled. End-labeling the purine strand of the duplex should confirm that the band being monitored corresponds to the triplex because it is possible that the labeled-ODN could be involved in a strand displacement and form a duplex with the purine rich top strand in ODN-A and-B.

To obtain another measure of the rate of triplex formation, the length of the incubation of the third strand with the duplex at 4°C can be varied: 1,3,6,9, 12 and 24 h. The ratio of triplex to ODN bands as a function of incubation time (using 32P-labeled ODN and phosphorimaging technology) should provide rate data.

Measuring the relative intensities of the triplex and ODN bands gives the triplex fraction (q). Using the following concentrations of the labeled ODN (0,0.05, 0.10,0.25,0.5,0.75 SM using 20 yM duplex), it should be possible to calculate a binding rate as follows: d DUPLEX/dt = d ODN/dt =-d [TRIPLEX]/dt =- k DUPLEX ODN + k TRIPLEX because kon >>> koff (Maher, L. J., III, Dervan, P. D. and Wold, B. (1990) Biochemistry 29,8820-8826) and because there is an excess of the DUPLEX, the second order rate constant can be formulated as a pseudo-first order rate equation: d ODN/dt =-K ODN; where K =-kon DUPLEX.

Integration of this equation gives: ln ODN/ ODN. = Kt; where ODN o is the initial ODN concentration that can form TRIPLEX.

This basically translates to: K = {In ( ODN o- TRIPLEX)/ ODN o}/t with the half life for the association, i. e., tl/2 = 0.693/K.

To measure triplex affinity, the duplex can be incubated for 24 h with different equivalents of the labeled third strand (unmodified and sidechain modified.

The same triplex fraction (q) can be calculated and presented as a function of ODN concentration to obtain binding constants.

Fidelity studies. It has been shown that the 5PENH2-dU sidechains do not affect Watson-Crick base pairing fidelity (Table 1 and Heystek et al., supra) using TM measurements. To ensure that the sidechains do not affect triplex formation, mismatches can be introduced between the third strand and the duplex: normally T binds to the A in an A: T pair and C to the G in a G: C pair. Duplexes-C and-D (see below) should serve to demonstrate that 5PENH2-dU (ODN-b) and 5P=NHz-dC (ODN-s) will not recognize T: A and C: G base pairs, respectively.

The ODNs (a and q) in Table B below, are to serve as controls. The TM's of the different potential triplexes can be determined and compared to those without a mismatch (duplex-A with ODN-a and duplex-B with ODN-q).

From the TM values, the ALG between the mismatch vs the normal sequence can be calculated.

TABLE B duplex 5'-TGAGAAAAAAATAAAAAAACCAA duplex 5'-TGAGAAAAAGACAGAGAAACCAA C 3'-ACTCTTTTTTTATTTTTTTGGTT D 3'-ACTCTTTTTCTGTCTCTTTGGTT ODN-a 5'-TTTTTTTTTTTTTTT ODN-q 5'-TTTTTCTCTCTCTTT ODN-b 5'-TTTTTTTXTTTTTTT ODN-s 5'-TTTTTXTXTCTCTTT Methylation protection studies. The occupancy of the major groove of the Watson-Crick duplex by a pyrimidine third strand involves Hoogsteen base pairing with the purine rich strand. If there are G: C base pairs in the Watson-Crick duplex, as there are in Duplex-B (Table A), the C in the third strand ODN should H-bond with the N7- G position. This causes the N7-G site, for steric reasons, to be unreactive to methylation by dimethyl sulfate (DMS), an event that can be followed using Maxam-Gilbert G-lane chemistry and denaturing PAGE (Maxam, A. M., and Gilbert, W. (1980) Methods Enzymol.

65,499-560). This approach can verify that there is a 3-strand structure and that the third strand occupies the major groove (Soyfer, V. N. and Potaman, V. N.

(1996) Triple-Helical Nucleic Acids, Springer-Verlag, New York).

The duplex target is 5'-end labeled on the top strand using T4 kinase and 32p y-ATP. Approximately 0.5 pmol of duplex is dissolved in the buffer described for the TM studies (see above), and 20 pmol of the third strand (unmodified or modified with the different sidechains) is added. The incubation is kept at room temperature overnight and then treated with dimethyl sulfate using standard Maxam-Gilbert G-lane conditions

(Maxam and Gilbert, supra). The resulting N7- methylguanine lesions are converted into single-strand breaks using hot piperidine and the breaks sequenced on a 20% denaturing PAGE gel. The Maxam-Gilbert G, G+A and C marker lanes are included for reference.

The presence of the triplex should block N7- methylguanine formation at the 4 G's that are within the central 15 base pair duplex target sequence, (see Table A). This should result in a loss of cleavage bands that correspond to G's in the triplex region. No change in 7-methylguanine should be observed outside of the triplex forming region.

The following example is provided to merely illustrate an embodiment of the invention and is not intended to limit the invention in any way.

EXAMPLE I A. Effect of 5-substituted-2'-deoxypyrimidine modification of duplex stability.

The TM'S of duplexes with 5- (3-aminopropyn-1-yl)-2'- deoxyuridine substitutions (5=PNH2-dU), 5- (3- aminopropyl)-2'-deoxyuridine (5P-NH2-dU) and 5- (3- hydroxypropyl)-2'-deoxyuridine (5P-OH-dU) are shown in Table I along with the sequences studied Heystek et al., supra). Oligomers with one or more 5PENH2-dU substitutions show a very significant increase in TM (Table 1). At 100 mM NaCl, a single substitution (ODN-2) resulted in a 4.5 °C increase, while an oligomer with four 5P=NH2-dU residues (ODN-3) showed an 11.9° increase (Table 1). This increase exceeded that observed with the neutral propyne sidechain (Wagner et al, supra) (Figure

1,5P=H-dU) by >1 °C per residue in the same sequence (ODN-4 and-5). The introduction of flexible 3- aminopropyl sidechains, 5P-NH2-dU (Figure 1, ODN-6) decreased the TM at 100 mM NaCl by 2.4 °C, and the presence of neutral 3-hydroxypropyl appendages, 5P-OH- dU, (ODN-7) resulted in a duplex structure that is 7 °C less stable (Table 1). These data with the flexible 5P- NH2-dU and 5P-OH-dU (Figure 1) sidechains are similar to those reported for 5-butane substituted nucleotides and indicate that any stabilizing electrostatic effect of the tethered cationic amine in 5P-NH2-dU is countered by the destabilizing effect of the aliphatic sidechain (Hashimoto et al., supra; Hashimoto, H., Nelson, M. G. and Switzer, C. (1993) J. Org. Chem. 58,4194-4195). In previous studies, 5- (m-N-aminoalkyl)-carbamoyl-2'- deoxyuridine substitutions have caused position- dependent increases in TM's, although the increase is < 1 °C per modification at 100 mM salt (Ueno, Y., Kumagi, I., Haginoya, N. and Matsuda, A. (1997) Nucleic Acids Res. 25,3777-3782; Haginoya, N.; Ono, A.; Nomura, Y.; Ueno, Y.; Matsuda, A. (1997) Bioconjugate Chem. 8,271- 280) Clearly the location of the cationic charge determines the magnitude of the stabilization. The 4 °C increase per 5PENH2-dU residue compares to-3° increase in TM when an oligomer with a neutral Rp- methylphosphonate residue is paired with its complementary natural oligodeoxynucleotide (Bower, M.; Summers, M. F., Powell, C., Shinozuka, K., Regan, J. B., Zon, G., Wilson, W. D. (1987) Nucleic Acids Res. 15, 4915-4930; Vyazovkina, E. V., Savchenko, E. V., Lokhov, S. G., Engels, J. W., Wickstrom, E. and Lebedev, A. V.

(1994) Nucleic Acids Res. 22,2404-2409). The Sp-isomer binds inefficiently to its complement (Lesnikowski, Z.

J., Jaworska, M., and Stec, W. J. (1988) Nucleic Acids Res. 18,2109-2115).

If the stabilization of duplex DNA by 5P=-NH2-dU substitution (s) has an electrostatic component, then the TM's of the oligomers should not be as sensitive as unmodified ODNs to ionic strength. The relationship between salt concentration vs TM for the different ODNs is shown in Table 1. As anticipated, the ATM over the range of NaCl concentration (50 to 500 mM) is smaller for ODN-3 (7.8 °C) vs unmodified duplex ODN-1 (13.6 °C).

Previous work showed that an ODN fully substituted with 5P-NH2-dU sidechains is completely insensitive to salt concentration (Hashimoto et al., supra).

To demonstrate the potential of the 5PENH2-dU modified nucleotides to stabilize RNA-DNA complexes, a chimeric 14mer was synthesized and the stability of duplexes with natural DNA (Table 1, ODN-11) and DNA containing four of the 5P=NH2-dU substitutions opposite the RNA bases (ODN-12) was measured. The TM data show that the 5PENH2-dU modification increases the melting of ODN-12 by 10.2 °C in comparison to unmodified ODN-11 (Table 1).

Table 1.

Stability of unmodified and sidechain modified duplex oligomers TM (°C) NdCl (mM)' ODN DUD1eXb 50 100 200 500 1 5'-TGTATAGGGAGAGAAAG-3'40. 7 44.0 50.0 54.3 3'-TCCCTCTCTTTC-5' 2 5'-TGTATAGGGAGAGAAAG-3'44.5 48.5 52.5 56.0 3'-TCCCTCXCXTTC-5' 3 5'-TGTATAGGGAGAGAAAG-3'52.5 55.9 57.5 60.3 3'-TCCCXCXCXTXC-5' 4 5'-TGTATAGGGAGAGAAAG-3'n. d. C 47. 2 n-d-n. d.

3'-TCCCTCPCTTTC-5' 5 5'-TGTATAGGGAGAGAAAG-3'n. d. 52.1 n. d. n. d.

3'-TCCCPCPCPTPC-5' 6 5'-TGTATAGGGAGAGAAAG-3'39.0 41.6 44.2 46.2 3'-TCCCYCYCYTYC-5' 7 5'-TGTATAGGGAGAGAAAG-3'34.7 37.0 40.1 45.3 3'-TCCCZCZCZTZC-5' 8 5'-TGTATAGGGAGTGAAAG-3'31.8 n. d. n. d. n. d.

3'-TCCCTCXCTTTC-5' 9 5'-TGTATAGGGAGGGAAAG-3'34.8 n. d. n. d. n. d.

3'-TCCCTCXCTTTC-5' 10 5'-TGTATAGGGAGCGAAAG-3'30.9 n. d. n. d. n. d.

3'-TCCCTCXCTTTC-5' 11 5'-AGCGGAAAAGCACC-n. d. 58.8 n. d. n. d.

3I d 3'-TCGCCTTTTCGTCC-5' 12 5'-AGCGGAAAAGCACC-3'n. d. 69.0 n. d. n.. d 3'-TCGCCXXXXCGTCC-5' a Temperature ramped from 15 to 80°C at a rate of 1 °/min and UV monitored at 260 and 280 nm. Thermal TM'S were calculated using the first derivative method.

Conditions: 2.5 UM duplex, 10 mM sodium phosphate buffer (pH 7.0), 0.1 mM EDTA and NaCl from 50-500 mM as indicated. b See Figure 1 for structures of X = 5PNH2- dU, P = 5PH-dU, Y = 5P-NH2-dU and Z = 5P-OH-dU; c n. d.

= not determined; d italicized A in ODNs-11 and-12 are RNA adenosine residues..

B. Base pairing fidelity of 5P=NH2-dU modification.

To confirm that the fidelity of base pairing was not compromised by the 5PENH2-dU sidechain, the effect on duplex stability of the three potential mismatches opposite the modified residue was measured. The studies (Table 1, ODNs 8-10) show that the TM drops by 10-14 °C upon introduction of a mismatch opposite 5PENH2-dU, which is in the range of that observed for mismatches involving thymine residues: the AT, ls for the mismatches in the unmodified oligomers are: T-T, 13.4 °C; T-G, 10.2 °C; and T-C, 7.1 °C.

C. Effect of 5-substituted-2'-deoxypyrimidine modification of triplex stability.

TM studies. Studies have been performed to determine how the cationic sidechains affect triplex formation using thermal denaturation and gel mobility assays. To date, triplex structures with one or two 5P= NH2-dU and 5P-OH-dU modifications have been analyzed (Table 2). The results are very similar to that observed with duplex formation: the rigid aminopropynyl sidechain (Table 2, ODNs 14 and 15) stabilizes formation of the 3-stranded complex by >5 °C per residue, while the flexible hydroxypropyl sidechain (Table 2, ODNs 16 and 17) destabilzes the triplex relative to an unmodified Ti. strand. Based on literature reports, the neutral 5P H-dU sidechain stabilizes triplex formation, but the increase in triplex TM is 2.4 °C per residue (Froehler et al., supra). In contrast to 5P=H-dU, the dC analogue destabilizes triplex formation by ~ 3. 4 °C per residue

(Froehler et al., supra). It appears that the propyne group reduces the pKa for protonation of the N3-C position to-3.5 vs. 4.4 for 5-methyl-dC (Froehler et al., supra).

Since the pKa's of CH3CH2CH2NH2, CH2=CHCH2NH2 and CH=CCH2NH2 are 10.7,9.5 and 8.2, respectively, there is extensive e- withdrawal from the amino group into the alkyne pi system.

Therefore, the terminal amino group on the sidechain of 5P=NH2-dC could moderate the e-withdrawal of the propyne group from the aromatic heterocyclic base.

Table 2.

Stability of unmodified and sidechain modified triplex oligomersa oligo sequence° triplex TM duplex TK -mer (°C) (°C) 5'-d (TGAGTGAGTAAAAAAAAAAAAAAAGAGTGCCAA)-3' 13 3'-d (ACTCACTCATTTTTTTTTTTTTTTCTCACGGTT)-5'18.0 71.9 5'-d (TTTTTTTTTTTT)-3' 5'-d (TGAGTGAGTAAAAAAAAAAAAAAAGAGTGCCAA)-3' 14 3'-d (ACTCACTCATTTTTTTTTTTTTTTCTCACGGTT)-5' 25.1 72.0 5'-d (TTTTTTTXTTTTTTT)-3' 5t-d (TGAGTGAGTAAAAAAAAAAAAAAAGAGTGCCAA)-3' 15 3'-d (ACTCACTCATTTTTTTTTTTTTTTCTCACGGTT-5'29.3 72.0 5'-d (TTTTTXTTTXTTTTT)-3' 5'-d (TGAGTGAGTAAF'AGAGTGCCAA ?-3' 16 3'-d (ACTCACTCATTTTTTTTTTTTTTTCTCACGGTT)-5'n. d. ¢ 72.7 5'-d (TTTTTTTZTTTTTTT)-3' 5'-d (TGAGTGAGT GAGTGCCAA)-3' 17 3'-d (ACTCACTCATTTTTTTTTTTTTTTCTCACGGTT)-5'n. d. 71.9 5'-d (TTTTTZTTTZTTTTT)-3' a The duplex (2 HM, strand) was annealed in buffer containing 100 mM NaCl, 1 mM EDTA and 1 mM spermine (pH 7.0) from 90 °C to room temperature and third strand (2 UM, strand) was then introduced and the solution maintained at 4 °C overnight before running TM. b X = 5PENH2_dU; Z = 5P-OH-dU. ° n. d. = not detected.

Gel mobility studies-Triplex formation was partially verified using a 16% non-denaturing gel with buffer containing 100 mM NaCl, 10 mM Na- phosphate, 1 mM EDTA and 1 mM spermine (pH 7.0). The gel was run in a cold room (7 °C) at 50 V for 100 hours. The conditions used (not shown) prevented full resolution of the triplex and duplex bands because the size of the Watson-Crick duplex used to bind the third strand was too large, i. e., it is difficult to resolve the 66 base pair duplex from the 81 base pair triplex. However, the data did tentatively indicate that a triplex was formed only with the 5P=-NH2-dU substitution (s). This study will be repeated with smaller duplex targets.

C. Orientation of-aminoalkyl and-aminoalkynyl sidechains on pyrimidines.

The position adopted by the tethered ammonium ion has been established using an electrostatic footprinting method and by molecular modeling (Liang, G., Encell, L., Switzer, C. and Gold, B. (1995) J.

Am. Chem. Soc. 117,10135-10136; Dande, P., Liang, G., Chen, F.-X., Roberts, C., Switzer, C. and Gold, B. (1997) Biochemistry 36,6024-6032). Both methods give the same answer. The flexible sidechains 5P-NH2- dU sidechains adopt a conformation that places it toward the 3'-residue and near the floor of the major groove (Figure 3, Y). A similar orientation is predicted for the 5P-OH-dU residue. In contrast, the rigid 5P=NH2-dU sidechain points toward the 5'-residue (see Figure 3, X) and can make a salt bridge with the non-bridging phosphate oxygen (Heystek et al., supra).

In summary, the foregoing results demonstrate that the introduction of rigid 3-aminopropyn-1-yl sidechains at the 5-position of deoxyuridine results in ODNs with a marked increase in duplex (DNA-DNA and RNA-DNA) and triplex stability. In the case of the duplexes there is no decrease in base pairing fidelity. This should also be the case in triplex formation. The 5PENH2-dU (and presumably 5PENH2-dC) modified oligomers thus comprise potential antigene molecules that have both in vitro and in vivo to regulate gene expression and to detect the presence or absence of particular target sequences.