NUCLEOBASE CONJUGATES WITH CATIONIC BACKBONE LINKERS

[0001] The present disclosure relates to, among other things, nucleoside, nucleotide, and polynucleotide compounds, compositions, kits and methods that may be useful for DNA sequencing, the formation anl29004d use of labeled polynucleotides, and/or other uses. [0002] The analysis of complex mixtures of polynucleotides is important in many biological applications. In many situations, it is necessary to separate components of such mixtures to detect target polynucleotides of interest, to determine relative amounts of different components, and to obtain nucleotide sequence information, for example. [0003] Electrophoresis provides a convenient tool for analyzing polynucleotides. Typically, polynucleotides can be separated on the basis of length, due to differences in electrophoretic mobility. For example, in a matrix such as crosslinked polyacrylamide, polynucleotides typically migrate at rates that are inversely proportional to polynucleotide length, due to size-dependent obstruction by the crosslinked matrix. In free solution, polynucleotides tend to migrate at substantially the same rates because of their substantially identical mass-to-charge ratios, so that it is difficult to distinguish different polynucleotides based on size alone. However, distinguishable electrophoretic mobilities can be obtained in free solution using polynucleotides that contain different charge/mass ratios, e.g., by attaching to the polynucleotides a polymer or other chemical entity having a charge/mass ratio that differs from that of the polynucleotides alone (e.g., see US Patent No. 5,470,705). [0004] When different polynucleotides can be separated based on different electrophoretic mobilities, detection can usually be accomplished using a single detectable label, such as a radioisotope or fluorophore. However, in complex mixtures or when different-sequence polynucleotides have similar or identical mobilities, it is preferable to use two or more detectable labels to distinguish different polynucleotides unambiguously. [0005] In DNA sequencing, it is conventional to use two or more (usually four) different fluorescent labels to distinguish sequencing fragments that terminate with one of the four standard nucleotide bases (A, C, G and T, or analogs thereof). Such labels are usually introduced into the sequencing fragments using suitably labeled extension primers (dye- primer method) or by performing primer extension in the presence of nonextendable nucleotides that contain unique labels (Sanger dideoxy terminator method). Electrophoresis of the labeled products generates ladders of fragments that can be detected on the basis of elution time or band position.

[0006] When polynucleotides are synthesized by enzyme-mediated incorporation of labeled nucleotides, mixtures can be produced that contain the desired labeled polynucleotide product(s) plus residual labeled nucleotides. Electrophoresis of such mixtures can produce separation profiles in which the labeled nucleotides (if they are negatively charged) and breakdown products thereof (sometimes called "dye blobs) co-migrate with negatively charged labeled polynucleotides. Comigration of dye blobs can interfere with the identification or quantification of particular polynucleotide fragments and can cause incomplete or erroneous sequence determinations.

[0007] A factor that contributes to the presence of dye blobs is the need to use high concentrations of labeled nucleotides relative to unlabeled nucleotides (e.g., a ratio of 50:1) due to the weaker binding affinities of polymerase enzymes for labeled nucleotides. Dye blobs can be removed or substantially reduced in amount by subjecting such reaction mixtures to purification by methods such as size-exclusion gel chromatography or ethanol precipitation. However, such purification methods can undesirably reduce the levels of smaller DNA fragments and also increase the overall time to obtain results. [0008] Accordingly, in some embodiments, the present disclosure provides labeled nucleotides that can be effectively incorporated into polynucleotides by polymerases or other enzymes. In some embodiments, the present disclosure provides labeled nucleotides that migrate more slowly in electrophoresis than the corresponding labeled forms of standard nucleotides such as ATP, CTP, GTP, and TTP, thereby allowing improved detection of polynucleotides that are obscured by comigrating labeled standard nucleotides. In some embodiments, the present disclosure provides labeled nucleotides that have a substantially net neutral or net positive charge under selected pH conditions so that they cannot co-migrate with negatively charged analytes such as ribo- or deoxyribopolynucleotides. [0009] In some embodiments, the present disclosure provides conjugates comprising a dye labeled nucleobase of the form: (1) B-L-D, wherein B is a nucleobase, L is a linker whose backbone comprises at least one imidazolium moiety, and D comprises at least one fluorescent dye, or (2) B-L1-D1-L2-D2, wherein B is a nucleobase, Ll and L2 are linkers such that at least one of Ll and L2 is a linker whose backbone comprises at least one imidazolium moiety, and Dl and D2 are members of an energy transfer pair, such that one of Dl and D2 is an energy donor capable of emitting energy at a first wavelength and the other of Dl and D2 is capable of absorbing the energy emitted from the donor and emitting energy at a second wavelength in response thereto.

[0010] In some embodiments, the dye-labeled nucleobase is of the form B-L-D. In some embodiments, the backbone of L comprises a total of one, two, three, four, five, six, or seven imidazolium moieties.

[0011] In some embodiments, the backbone of L comprises at least two, or at least three, or at least four, or at least five, or at least six, or at least seven imidazolium moieties.

[0012] In some embodiments, L comprises 4 to 50 chain atoms.

[0013] In some embodiments, D comprises at least one xanthene, rhodamine, fluorescein,

[8,9]benzophenoxazine, cyanine, phthalocyanine, squaraine, or bodipy dye.

[0014] In some embodiments, the labeled nucleobase is of the form: B-L1-D1-L2-D2.

[0015] In some embodiments, the backbone of Ll comprises a total of one, two, three, four, five, six, or seven imidazolium moieties.

[0016] In some embodiments, the backbone of Ll comprises at least two, or at least three, or at least four, or at least five, or at least six, or at least seven imidazolium moieties.

[0017] In some embodiments, Ll comprises 4 to 50 chain atoms.

[0018] In some embodiments, L2 comprises 4 to 50, or 4 to 30, or 4 to 20 chain atoms.

[0019] In some embodiments, the backbone of L2 does not comprise an imidazolium moiety.

[0020] In some embodiments, the backbone of L2 comprises at least one imidazolium moiety.

[0021] In some embodiments, the backbones of both Ll and L2 each comprise at least one imidazolium moiety.

[0022] In some embodiments, Ll and L2 taken together comprise a total of two, three, four, five, six, or seven imidazolium moieties.

[0023] In some embodiments, Ll and L2 taken together comprise at least two, or at least three, or at least four, or at least five, or at least six, or at least seven imidazolium moieties.

[0024] In some embodiments, at least one of Dl or D2 comprises a xanthene, rhodamine, fluorescein, [8,9]benzophenoxazήie, cyanine, phthalocyanine, or squaraine dye.

[0025] In some embodiments, Dl is a donor dye and D2 is an acceptor dye.

[0026] In some embodiments, B comprises adenine, 7-deazaadenine, 7-deaza-8- azaadenine, cytosine, guanine, 7-deazaguanine, 7-deaza-8-azaguanine, thymine, uracil, or inosine.

[0027] In some embodiments, the conjugate has a net positive charge at a pH of 7 or greater, or has a net positive charge al a pH of 8 or greater.

[0028] In some embodiments, labeled nucleoside triphosphates are provided comprising a conjugate that contains an imidazolium-containing linker moiety. [0029] In some embodiments, the labeled nucleoside triphosphate is not 3 '-extendable. [0030] In some embodiments, the labeled nucleoside triphosphate is a 2',3'- dideoxynucleotide or 3'-fluoro-2',3'-dideoxynucleotide.

[0031] In some embodiments, the labeled nucleoside triphosphate contains a 3'-hydroxyl group.

[0032] In some embodiments, polynucleotides are provided comprising a conjugate that contains an imidazolium-containing linker moiety.

[0033] In some embodiments, at least one said conjugate is contained in a 3 ¹ terminal nucleotide subunit.

[0034] In some embodiments, the polynucleotide comprises a 3 ¹ terminal nucleotide subunit that is not 3 '-extendable, such as a 2',3'-dideoxynucleotide or 3'-fluoro-2',3'- dideoxynucleotide.

[0035] In some embodiments, the 3' terminal nucleotide subunit contains a 3'-hydroxyl group.

[0036] In some embodiments, at least one said conjugate is contained in a non-terminal nucleotide subunit.

[0037] In some embodiments, mixtures are provided comprising a plurality of different polynucleotides, wherein at least one polynucleotide contains a conjugate as described herein.

[0038] In some embodiments, the mixture comprises a plurality of different polynucleotides that each comprises the same type of 3' terminal nucleotide subunit. [0039] In some embodiments, the mixture comprises four classes of polynucleotides, wherein the polynucleotides in each class terminate with a different type of 3' terminal nucleotide subunit that identifies the polynucleotides in that class, wherein at least one said polynucleotide comprises a conjugate as described herein.

[0040] hi some embodiments, mixtures are provided comprising at least one labeled nucleoside triphosphate comprising a nucleobase conjugate that contains an imidazolium- containing linker moiety, and one or more of the following components: a 3'-extendable primer, a polymerase enzyme, one or more 3'-extendable nucleoside triphosphates that do not comprise a said conjugate, and/or a buffering agent.

[0041] In some embodiments, kits are provided comprising at least one labeled nucleoside triphosphate comprising a nucleobase conjugate that contains an imidazolium-containing

linker moiety, and one or more of the following components: a 3'-extendable primer, a polymerase enzyme, one or more 3'-extendable nucleoside triphosphates that do not comprise a said conjugate, and/or a buffering agent.

[0042] In some embodiments, at least one labeled nucleoside triphosphate is 3'- extendable.

[0043] In some embodiments, at least one labeled nucleoside triphosphate is not 3'- extendable.

[0044] In some embodiments, the kit or mixture comprises four different labeled nucleoside triphosphates that are complementary to A, C, T and G, such that at least one of the labeled nucleoside triphosphates comprises a nucleobase conjugate that contains an imidazolium-containing linker moiety.

[0045] In some embodiments, the four different labeled nucleoside triphosphates each comprise the same donor.

[0046] In some embodiments, the four different labeled nucleoside triphosphates are not 3 '-extendable.

[0047] In some embodiments, the four different labeled nucleoside triphosphates are 3'- extendable ribonucleoside triphosphates.

[0048] In some embodiments, at least one donor dye is an ortho-carboxyfluorescein. [0049] In some embodiments, the present disclosure provides a method of forming a labeled polynucleotide comprising reacting a first polynucleotide with a labeled nucleoside triphosphate comprising a conjugate as described herein in the presence of a primer extension reagent under conditions effective to form a modified polynucleotide containing at least one labeled nucleoside subunit from the labeled nucleoside triphosphate. [0050] In some embodiments, said reacting is performed in the presence of a template nucleic acid to which the first polynucleotide is complementary, and the primer extension reagent comprises a template-dependent polymerase.

[0051] In some embodiments, following said reacting, the modified polynucleotide is subjected to electrophoresis.

[0052] In some embodiments, the modified polynucleotide is subjected to electrophoresis without prior removal of residual labeled nucleoside triphosphate.

[0053] In some embodiments, methods comprise forming one or more labeled different- sequence polynucleotides, wherein at least one different-sequence polynucleotide contains a unique conjugate of the type described herein.

[0054] In some embodiments, one or more labeled different-sequence polynucleotides are separated by electrophoresis so as to separate different-sequence polynucleotides on the basis of size, and different-sequence polynucleotides are identified on the basis of electrophoretic mobility and, optionally, fluorescence signal,

[0055] In some embodiments, methods comprise forming four classes of polynucleotides which are complementary to a target polynucleotide sequence, by template-dependent primer extension, wherein the polynucleotides in each class terminate with a different terminator subunit type, and at least one polynucleotide contains a conjugate as described herein, separating the polynucleotides of the four classes on the basis of size to obtain a mobility pattern, and determining the sequence of the target polynucleotide sequence from the mobility pattern.

[0056] In some embodiments, terminator subunits are nonextendable.

[0057] In some embodiments, terminator subunits contain a 3'-hydroxyl group.

[0058] In some embodiments, at least one said conjugate has a net neutral or net positive charge at pH 7 or at pH 8.

[0059] The term "detectable label" refers to any moiety that, when attached to the compounds of the present teachings, renders such compounds detectable using known detection means. Exemplary detectable labels include but are not limited to fluorophores, chromophores, radioisotopes, spin-labels, enzyme labels, chemiluminescent labels that are detectable using a suitable detector or detection means, or a binding pair, for example, a ligand, such as an antigen or biotin, that can bind specifically with high affinity to a detectable anti-ligand, such as a labeled antibody or avidin. In some embodiments the labels can be fluorescent dyes such as fluorescein, rhodamine, cyanine, or pyrene dyes, for example.

[0060] "Enzymatically extendable" or "3 ¹ extendable" means a nucleotide or polynucleotide that is capable of being appended to a nucleotide or polynucleotide by enzyme action. A polynucleotide containing a 3' hydroxyl group is an example of an enzymatically extendable polynucleotide,

[0061] "Enzymatically incorporatable" means that a nucleotide is capable of being enzymatically incorporated onto the terminus, e.g. 3' terminus, of a polynucleotide chain, or internally through nick-translation of a polynucleotide chain, through action of a template- dependent or template-independent polymerase enzyme. A nucleotide-5' -triphosphate is an example of an enzymatically incorporatable nucleotide.

[0062] "Backbone-imidazolium linker moiety" refers to a moiety that comprises one or more imidazolium moieties in the backbone chain of atoms of a linker.

[0063] "Linker" refers to a moiety that links a dye to a substrate such as an oligonucleotide, or links one dye to another dye (e.g., links a donor to an acceptor dye). [0064] "Nucleobase" means a nitrogen-containing heterocyclic moiety capable of forming Watson-Crick type hydrogen bonds with a complementary nucleobase or nucleobase analog, e.g. a purine, a 7-deazapurine, or a pyrimidine. Typical nucleobases are the naturally occurring nucleobases adenine, guanine, cytosine, uracil, thymine, and analogs of naturally occurring nucleobases, e.g. 7-deazaadenine, 7-deaza-8-azaadenine, 7-deazaguanine, 7-deaza-8- azaguanine, inosine, nebularine, nitropyrrole, nitroindole, 2-amino-purine, 2,6-diamino- purine, hypoxanthine, pseudouridine, pseudocytidine, pseudoisocytidine, 5-propynyl- cytidine, isocytidine, isoguanine, 2-thiopyrimidine, 6-thioguanine, 4-thiothymine, 4- thiouracil, (/-methylguanine, //-methyl-adenine, <y-methylthymine, 5,6-dihydrothymine, 5,6-dihydrouracil, 4-methylindole, and ethenoadenine (Fasman, Practical Handbook of Biochemistry and Molecular Biology, pp. 385-394, CRC Press, Boca Raton, Fl (1989)). [0065] "Nucleoside" means a compound comprising a nucleobase linked to a C-Y carbon of a ribose sugar or sugar analog thereof. The ribose or analog may be substituted or unsubstituted. Substituted ribose sugars include, but are not limited to, those riboses in which one or more of the carbon atoms, preferably the 3 '-carbon atom, is substituted with one or more of the same or different substituents such as -R, -OR, -NRR or halogen (e.g., fluoro, chloro, bromo, or iodo), where each R group is independently -H, Ci-Cβ alkyl or C ₃ -C ₁₄ aryl. Particularly preferred riboses are ribose, 2'-deoxyribose, 2',3'-dideoxyribose _> 3'-haloribose (such as 3'-fluororibose or 3'-chlororibose) and 3'-alkylribose. Typically, when the nucleobase is A or G, the ribose sugar is attached to the N ⁹ -position of the nucleobase. When the nucleobase is C, T or U, the pentose sugar is attached to the N '-position of the nucleobase (Kornberg and Baker, DNA Replication, 2 ^nd Ed., Freeman, San Francisco, CA, (1992)). Other examples of sugar analogs include, but are not limited to, substituted or unsubstituted furanoses having more or fewer than 5 ring atoms, e.g., erythroses and hexoses and substituted or unsubstituted 3-6 carbon acyclic sugars. Typical substituted furanoses and acyclic sugars are those in which one or more of the carbon atoms are substituted with one or more of the same or different -R, -OR, -NRR or halogen groups, where each R is independently -H, (Ci-Ce) alkyl or (C ₁ -C ₁₄ ) aryl. Examples of substituted furanoses having 5 ring atoms include but are not limited to 2-deoxyribose, 2'-(Ci-C ₆ )alkylribose, 2'-(Ci- C ₆ )alkoxyribose (e.g., 2'-O-methyl ribose), 2'-(C ₅ -Ci ₄ )aryloxyribose, 2',3'-dideoxyribose, 2',3 -didehydroribose, 2'-deoxy-3'-haloribose, 2'-deoxy-3'-fluororibose, 2'-deoxy-3'- chlororibose, 2'-deoxy-3'- amino-ribose, 2'-deoxy-3'-(Ci-C,s)alkylribose, 2'-deoxy-3'-(Ci-

C ₆ )alkoxyribose, 2'-deoxy-3 ^t -(C ₅ -C ₁ 4)aryloxyribose, 3'-(Ci-C ₆ )aIkylribose-5'-triphosphate, 2'-deoxy-3'-(Cj-C ₆ )alkylribose -5 '-triphosphate, 2'-deoxy-3'-(C ₁ -C ₆ )alkoxyribose-5'- triphosphate, 2 ^t -deoxy-3 ^l -(C5-C ₁₄ )aryl-oxyribose-5'-triphosphate,

2'-deoxy-3'-haloribose-5'-triphosphate, ^-deoxy-S'-aminoribose -5'-triphosphate,

2',3'-dideoxyribose-5'-triphosphate or 2 ^t ,3'-didehydroribose-5'-triphosphate. Further sugar analogs include but are not limited to, locked nucleic acids such as

(e.g., see Wengel, et al. WO 99/14226, incorporated herein by reference).

[0066] "Nucleotide" means a phosphate ester of a nucleoside, either as an independent monomer or as a subunit within a polynucleotide. Nucleotide triphosphates (also referred to herein as nucleoside triphosphates) are sometimes denoted as "NTP", "dNTP" (2'-deoxypentose) or "ddNTP" (2\3'-dideoxypentose) to particularly point out the structural features of the ribose sugar. "Nucleotide 5'-triphosphate" refers to a nucleotide with a triphosphate ester group at the 5' position. The triphosphate ester group may include sulfur substitutions for one or more phosphate oxygen atoms, e.g. α-thionucleotide 5'-triphosphates.

[0067] "Nonextendable" or "3 ¹ nonextendable" refers to the fact that a terminator is incapable, or substantially incapable, of being extended in the 3' direction by a template- dependent DNA or RNA polymerase.

[0068] "Nucleotide subunit" or "polynucleotide subunit" refers to a single nucleotide or nucleotide analog within a polynucleotide or polynucleotide analog.

[0069] "Polynucleotide" refers to linear polymers of natural nucleotide monomers or analogs thereof, including for example, double- and single-stranded deoxyribonucleotides, ribonucleotides, α-anomeric forms thereof, and the like. A polynucleotide may be composed entirely of deoxyribonucleotides, ribonucleotides, or analogs thereof, or may contain blocks or mixtures of two or more different monomer types. Usually nucleoside monomers are linked by phosphodiester linkages. However, polynucleotides containing non-phosphodiester linkages are also contemplated. "Polynucleotide" also encompasses polymers that contain one or more non-naturally occurring monomers and/or intersubunit linkages, such as peptide nucleic acids (PNAs, e.g., polymers comprising a backbone of amide-linked N-(2-aminoethyl)-glycine subunits to which nucleobases are attached via the non-amide backbone nitrogens. See Nielsen

et al., Science 254:1497-1500 (1991)). Polynucleotides typically range in size from a few monomelic units, e.g. 8-40, to several thousand monomeric units. Whenever a polynucleotide is represented by a sequence of letters, such as "ATGCCTG," it will be understood that the nucleotides are in 5'->3' order from left to right and that "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine, and "T" denotes thymidine, unless otherwise noted.

[0070] "Phosphate analog" refers to an analog of phosphate wherein one or more of the oxygen atoms is replaced with a non-oxygen moiety. Exemplary phosphate analogs including phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoro- anilothioate, phosphotriester, phosphoranilidate, phosphoramidate, alkylphosphonates such as methylphosphonates, boronophosphates.

[0071] "Spectrally resolvable" means that two or more dyes have emission bands that are sufficiently distinct, i.e., sufficiently non-overlapping, that they can be distinguished on the basis of a unique fluorescent signal generated by each dye.

[0072] "Template nucleic acid" refers to any nucleic acid or polynucleotide that is capable of annealing with a primer polynucleotide. Exemplary template nucleic acids include DNA, KNA, which DNA or RNA may be single stranded or double stranded. More particularly, template nucleic acid may be genomic DNA, messenger RNA, cDNA, DNA amplification products from a PCR reaction, and the like. Methods for preparation of template DNA may be found elsewhere (ABI PRISM™ Dye Primer Cycle Sequencing Core Kit). [0073] "Terminator" means an enzymatically incorporatable nucleotide which prevents subsequent incorporation of nucleotides to the resulting polynucleotide chain and thereby halts polymerase-mediated extension. Typical terminators lack a 3'-hydroxyl substituent and include 2\3'-dideoxyribose, 2',3'-didehydroribose, and 2 ^l ,3'-dideoxy-3'-haloribose, e.g. 3'-deoxy-3'- fluoro-ribose or 2 ¹ ,3'-dideoxy-3'-fluororibose, for example. Alternatively, a ribofuranose analog can be used, such as 2',3'-dideoxy-β-D-ribofuranosyl, β-D-arabinofuranosyl, 3'- deoxy-β-D-arabinofuranosyl, 3'-amino~2\3 ^> -dideoxy-β-D-ribofuranosyl, and 2',3'-dideoxy- 3'-fluoro-β-D-ribofuranosyl (see, for example, Chidgeavadze et al., Nucleic Acids Res., 12: 1671-1686 (1984), and Chidgeavadze et al. FEB. Lett, 183: 275-278 (1985)). Nucleotide terminators also include reversible nucleotide terminators (Metzker et al. Nucleic Acids Res., 22(20):42S9 (1994)).

[0074] Generally, whenever a compound mentioned in this disclosure contains a positive or negative charge, it should be understood that such compound may also be accompanied by a

9 SUBSTTTUTE SHEET (RULE 26)

suitable counterfoil that balances the positive or negative charge. Exemplary positively charged counterions include, without limitation, H ⁺ , NH ₄ ⁺ , Na ⁺ , K ⁺ , Mg ²⁺ , trialkylammonium (such as triethylammonium), tetraalkylammonium (such as tetraethyiammonium), and the like. Exemplary negatively charged counterions include, without limitation, carbonate, bicarbonate, acetate, chloride, and phosphate, for example. Also, although particular resonance structures may be shown herein, such structures are intended to include all other possible resonance structures.

[0075] In some aspects, the present invention provides compositions that comprise at least one dye-labeled nucleobase of the type described herein. Such compositions include not only nucleobase-dye conjugates as independent molecules, but also as nucleosides, nucleotides and polynucleotides containing such conjugates, as well as mixtures, solids, or solutions containing any of the foregoing.

[0076] In some embodiments, a dye-labeled nucleobase of the invention has the form B-L- D, wherein B is a nucleobase, L is a linker whose backbone comprises at least one imidazolium moiety, and D comprises at least one fluorescent dye.

[0077] Nucleobase B may be any moiety capable of forming Watson-Crick hydrogen bonds with a complementary nucleobase or nucleobase analog, as set forth above. Typically, B is a nitrogen-containing heterocyclic moiety such as a 7-deazapurine, purine, or pyrimidine nucleotide base. In certain embodiments, B is uracil, cytosine, 7-deazaadenine, or 7- deazaguanosine. When B is a purine, the linker is usually attached to the 8-position of the purine. When B is a 7-deazapurine, the linker to the dye is usually attached to the 7-position of the 7-deazapurine. When B is pyrimidine, the linker is usually attached to the 5-position of the pyrimidine.

[0078] Fluorescent dye D may be any fluorescent dye that is suitable for the purposes of the invention. Typically, the fluorescent dye comprises a resonance-delocalized system or aromatic ring system that absorbs light at a first wavelength and emits fluorescent light at a second wavelength in response to the absorption event, A wide variety of such dye molecules are known in the art. For example, fluorescent dyes can be selected from any of a variety of classes of fluorescent compounds, such as xanthenes, rhodamines, fluoresceins, cyanines, phthalocyanines, squaraines, and bodipy dyes.

[0079] In some embodiments, the dye comprises a xanthene-type dye, which contains a fused three-ring system of the form:

This parent xanthene ring may be unsubstituted (i.e., all substituents are H) or may be substituted with one or more of a variety of the same or different substituents, such as described below.

[0080] In some embodiments, the dye contains a parent xanthene ring having the general structure:

[0081] In the parent xanthene ring depicted above, A ¹ is OH or NH ₂ and A ² is O or NH ₂ ⁺ . When A ¹ is OH and A ² is O, the parent xanthene ring is a fluorescein-type xanthene ring. When A ¹ is NH ₂ and A ² is NH ₂ ⁺ , the parent xanthene ring is a rhodamine-type xanthene ring. When A ¹ is NH ₂ and A ² is O, the parent xanthene ring is a rhodol-type xanthene ring. In the parent xanthene ring depicted above, one or both nitrogens of A ¹ and A ² (when present) and/or one or more of the carbon atoms at positions C-I, C-2, C-4, C-5, C-I, C-8 and C-9 can be independently substituted with a wide variety of the same or different substituents. In some embodiments, typical substituents include, but are not limited to, -X, -R, -OR, -SR, - NRR, perhalp (Ci-C ₆ ) alky!,-CX ₃ , -CF ₃ , -CN, -OCN, -SCN, -NCO, -NCS, -NO, -NO ₂ , - N ₃ , -S(O) ₂ O ^" , -S(O) ₂ OH, -S(O) ₂ R, -C(O)R, -C(O)X, -C(S)R, -C(S)X, -C(O)OR, -C(O)O ^" , -C(S)OR, -C(O)SR, -C(S)SR, -C(O)NRR, -C(S)NRR and -C(NR)NRR, where each X is independently a halogen (preferably -F or Cl) and each R is independently hydrogen, (Ci -C ₆ ) alkyl, (Ci-C ₆ ) alkanyl, (Ci-C ₆ ) alkenyl, (Ci-C ₆ ) alkynyl, (C ₅ -C ₂₀ ) aryl, (C ₆ -C ₂₆ ) arylalkyl, (C ₅ - C ₂ o) arylaryl, heteroaryl, 6-26 membered heteroarylalkyl 5-20 membered heteroaryl- heteroaryl, carboxyl, acetyl, sulfonyl, sulfinyl, sulfone, phosphate, or phosphonate. Moreover, the C-I and C-2 substituents and/or the C-I and C-8 substituents can be taken together to form substituted or unsubstituted buta[l,3]dieno or (Cs-C ₂ o) aryleno bridges. Generally, substituents which do not tend to quench the fluorescence of the parent xanthene ring are preferred, but in some embodiments quenching substituents may be desirable. Substituents that tend to quench fluorescence of parent xanthene rings are electron- withdrawing groups, such as -NO2, -Br, and -I. In some embodiments, C-9 is unsubstituted.

In some embodiments, C-9 is substituted with a phenyl group. In some embodiments, C-9 is substituted with a substituent other than phenyl.

[0082] When A ¹ is NH ₂ and/or A ² is NH ₂ ⁺ , these nitrogens can be included in one or more bridges involving the same nitrogen atom or adjacent carbon atoms, e.g., (Ci-C ₁₂ ) alkyldiyl,

(Ci-Ci ₂ ) alkyleno, 2-12 membered heteroalkyldiyl and/or 2-12 membered heteroalkyleno bridges.

[0083] Any of the substituents on carbons C-I, C-2, C-4, C-5, C-7, C-8, C-9 and/or nitrogen atoms at C-3 and/or C-6 (when present) can be further substituted with one or more of the same or different substituents, which are typically selected from -X, -R', =0, -OR', -

SR', =S, -NR ¹ R', =NR', -CX ₃ , -CN, -OCN, -SCN, -NCO, -NCS, -NO, -NO ₂ , =N ₂ , -N ₃ , -

NHOH, -S(O) ₂ O ^" , -S(O) ₂ OH, -S(O) ₂ R ¹ , -P(0)(0 ^" ) ₂ , -P(O)(OH) ₂ , -C(O)R ¹ , -C(O)X, -C(S)R', -

C(S)X, -C(O)OR', -C(O)O-, -C(S)OR', -C(O)SR', -C(S)SR ¹ , -C(O)NR ¹ R ¹ , -C(S)NR ¹ R' and -

C(NR)NR ¹ R', where each X is independently a halogen (preferably -F or -Cl) and each R' is independently hydrogen, (Ci-Cβ) alkyl, 2-6 membered heteroalkyl, (Cs-C] ₄ ) aryl or heteroaryl, carboxyl, acetyl, sulfonyl, sulfinyl, sulfone, phosphate, or phosphonate.

[0084] Exemplary parent xanthene rings include, but are not limited to, rhodamine-type parent xanthene rings and fiuorescein-type parent xanthene rings.

[0085] In some embodiments, the dye contains a rhodamine-type xanthene dye that includes the following ring system:

[0086] In the rhodamine-type xanthene ring depicted above, one or both nitrogens and/or one or more of the carbons at positions C-I, C-2, C-4, C-5, C-7 or C-8 can be independently substituted with a wide variety of the same or different substituents, as described above for the parent xanthene rings, for example. Exemplary rhodamine-type xanthene dyes include, but are not limited to, the xanthene rings of the rhodamine dyes described in US Patents 5,936,087, 5,750,409, 5,366,860, 5,231,191, 5,840,999, 5,847,162, and 6,080,852 (Lee et al.), PCT Publications WO 97/36960 and WO 99/27020, Sauer et al, J. Fluorescence 5(3):247-261 (1995), Arden-Jacob, Neue Lanwellige Xanthen-Farbstoffe βr Fluoreszenzsonden und Farbstoff Laser ; Verlag Shaker, Germany (1993), and Lee et al., Nucl. Acids Res. 20:2471-2483 (1992).

[0087] In some embodiments, the dye comprises a fluorescein-type parent xanthene ring having the structure:

[0088] In the fluorescein-type parent xanthene ring depicted above, one or more of the carbons at positions C-I, C-2, C-4, C-S, C-7, C-8 and C-9 can be independently substituted with a wide variety of the same or different substituents, as described above for the parent xanthene rings. Exemplary fluorescein-type parent xanthene rings include, but are not limited to, the xanthene rings of the fluorescein dyes described in US Patents 4,439,356, 4,481,136, 5,188,934, 5,654,442, and 5,840,999, WO 99/16832, and EP 050684. Also included within the scope of "fluorescein-type parent xanthene ring" are the extended xanthene rings of the fluorescein dyes described in US Patents 5,750,409 and 5,066,580. [0089] In some embodiments, the dye comprises a rhodamine dye, which comprises a rhodamine-type xanthene ring in which the C-9 carbon atom is substituted with an orthocarboxy phenyl substituent (pendent phenyl group). Such compounds are also referred to herein as orthocarboxyfluoresceins. A particularly preferred subset of rhodamine dyes are 4,7,-dichlororhodamines. Typical rhodamine dyes include, but are not limited to, rhodamine B, 5-carboxyrhodamine, rhodamine X (ROX), 4,7-dichlororhodamine X (dROX), rhodamine 6G (R6G), 4,7-dichlororhodamine 6G, rhodamine 110 (RI lO), 4,7-dichlororhodamine 110 (dRHO), tetramethyl rhodamine (TAMRA) and 4,7-dichloro-tetramethylrhodamine (dTAMRA). Additional rhodamine dyes can be found, for example, in US Patents 5,366,860 (Bergot et al.), 5,847,162 (Lee et al.), 6,017,712 (Lee et al.), 6,025,505 (Lee et al.), 6,080,852 (Ue et al.), 5,936,087 (Benson et al.), 6,111,116 (Benson et al.), 6,051,719 (Benson et al.), 5,750,409, 5,366,860, 5,231,191, 5,840,999, and 5,847,162, US application Serial No. 09/325,243 filed June 3, 1999, PCT Publications WO 97/36960 and WO 99/27020, Sauer et al, 1995, J. Fluorescence 5(3):247-261, Arden-Jacob, 1993, Newe Lanwellige Xanthen- Farbstoffe fiir Fluoresenzsonden und Farbstoff Laser, Verlag Shaker, Germany, and Lee et al, Nucl. Acids Res. 20(10):2471-2483 (1992), Lee et al., Nucl. Acids Res. 25:2816-2822 (1997), and Rosenblum et al., Nucl Acids Res. 25:4500-4504 (1997), for example. In some embodiments, the dye is a 4,7-dichloro-orthocarboxyrhodamine.

[0090] In some embodiments, the dye comprises a fluorescein dye, which comprises a fluorescein-type xanthene ring in which the C-9 carbon atom is substituted with an

orthocarboxy phenyl substituent (pendent phenyl group). A preferred subset of fluorescein- type dyes are 4,7,-dichlorofluoresceins. Typical fluorescein dyes include, but are not limited to, 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM). Additional typical fluorescein dyes can be found, for example, in US Patents 5,750,409, 5,066,580, 4,439,356, 4,481,136, 5,188,934 (Menchen et al.), 5,654,442 (Menchen et al.), 6,008,379 (Benson et al.), and 5,840,999, PCT publication WO 99/16832, and EPO Publication 050684. In some embodiments, the dye is a 4,7-dichloro-orthocarboxyfluorescein.

[0091] In some embodiments, the dye can be a cyanine, phthalocyanine, squaraine, or bodipy dye, such as described in the following references and references cited therein: Patent No. 5,863,727 (Lee et al.), 5,800,996 (Lee et al.), 5,945,526 (Lee et al.), 6,080,868 (Lee et al.), 5,436,134 (Haugland et al.), US 5,863,753 (Haugland et al.), 6,005,113 (Wu et al.), and WO 96/04405 (Glazer et al.).

[0092] Rhodamine dyes for use in connection with the present teachings can include, for example, a rhodamine dye having the structure:

where R _I -R _O are each independently selected from -H, -F, -Cl, -Br, -I, -CN, -CO ₂ H, -CO ₂ X, - CO ₂ R, -SO ₃ H, -SO ₃ X, -SO ₃ R, halogen, C]-Ci ₀ alkyl, Ci-Ci ₀ alkenyl, Ci-Ci ₀ alkynyl, Ci-C ₁₀ alkoxy, Ci-Ci ₀ alkylamine, Ci-Cio mercaptyl, Cj-Cio alkylsulfonate, C ₃ -Ci ₀ cycloalkyl, Gr Cio cycloalkenyl, C ₃ -Ci ₀ heterocyclic, C ₃ -Ci _O aromatic, Cs-C ₆ heteroaromatic, where each alkyl, alkenyl, alkynyl, alkoxy, alkylamine, mercaptyl, alkylsulfonate, cycloalkyl, cycloalkenyl, heterocyclic, aromatic and heteroaromatic is optionally further substituted by F, Cl, Br, I, -CO ₂ H, -CO ₂ X, -CO ₂ R, -SO ₃ H, -SO ₃ X or -SO ₃ R where X is a counterion and R is Ci-C ₆ alkyl,

[0093] Rg-Rn are each independently selected from Ci-C ₁₀ alkyl, Ci-Cio alkenyl, Q-Cio alkynyl, Ci-Ci ₀ alkoxy, Ci-Cio alkylamine, Cj-Cio mercaptyl, Ci-Ci ₀ alkylsulfonate, C ₃ -Ci ₀ cycloalkyl, C ₄ -Ci _O cycloalkenyl, C ₃ -Ci _O aromatic, benzyl, benzoyl, biphenyl where each alkyl, alkenyl, alkynyl, alkoxy, alkylamine, mercaptyl, alkylsulfonate, cycloalkyl,

cycloalkenyl, aromatic, benzyl, benzoyl and biphenyl is optionally further substituted by F,

Cl, Br, I, -CO ₂ H, -CO ₂ X, -CO ₂ R, -SO ₃ H, -SO ₃ X or -SO ₃ R where X is a counterion and R is

Ci-C ₆ alkyl,

[0094] Ri taken together with R? forms a 5-7 membered ring that is saturated or unsaturated, and is optionally substituted by one or more C ₁ -C ₆ alkyl, Ci-C ₆ alkylamine or

C ₁ -C ₆ alkylsulfonate moieties,

[0095] R ₂ taken together with Rj ₀ forms a 5-7 membered ring that is saturated or unsaturated, and is optionally substituted by one or more C)-C ₆ alkyl, Ci-C ₆ alkylamine or

Cj-C ₆ alkylsulfonate moieties,

[0096] R ₃ taken together with R ₄ forms a benzo or naphtha ring optionally substituted by one or more of -F, -Cl, -Br, -I, -CN, -CO ₂ H, -CO ₂ X, -CO ₂ R, -SO ₃ H, -SO ₃ X, -SO ₃ R, halogen,

Ci-C ₁ O alkyl, Ci-Cio alkenyl, CJ-CJO alkynyl, Cj-Cio alkoxy, Ci-Cio alkylamine, Ci-Cio mercaptyl, Ci-C] ₀ alkylsulfonate, C ₃ -Ci _O cycloalkyl, C ₄ -Ci ₀ cycloalkenyl, C ₃ -CiO heterocyclic, C ₃ -C ₁₀ aromatic, Cs-C ₆ heteroaromatic, where each alkyl, alkenyl, alkynyl, alkoxy, alkylamine, mercaptyl, alkylsulfonate, cycloalkyl, cycloalkenyl, heterocyclic, aromatic and heteroaromatic is optionally further substituted by F, Cl, Br, I, -0-, -S-, -NH-, -

NR- -CO ₂ H, -CO ₂ X, -CO ₂ R, -SO ₃ H, -SO ₃ X or -SO ₃ R where X is a counterion and R is Ci-C ₆ alkyl,

[0097] R ₅ taken together with R ₆ forms a benzo or naphtha ring optionally substituted by one or more of -F, -Cl, -Br, -I, -CN, -CO ₂ H, -CO ₂ X, -CO ₂ R, -SO ₃ H, -SO ₃ X, -SO ₃ R, halogen,

Ci-Cio alkyl, CpC] ₀ alkenyl, Cj-Cio alkynyl, C ₁ -Q0 alkoxy, Ci-Ci ₀ alkylamine, Ci-Ci ₀ mercaptyl, C]-C] ₀ alkylsulfonate, C ₃ -Cj ₀ cycloalkyl, C ₄ -Ci ₀ cycloalkenyl, C ₃ -C) ₀ heterocyclic, C ₃ -Cj _O aromatic, Cs-C ₆ heteroaromatic, where each alkyl, alkenyl, alkynyl, alkoxy, alkylamine, mercaptyl, alkylsulfonate, cycloalkyl, cycloalkenyl, heterocyclic, aromatic and heteroaromatic is optionally further substituted by F, Cl, Br, I, -CO ₂ H, -CO ₂ X, -

CO ₂ R, -SO ₃ H, -SO ₃ X or -SO ₃ R where X is a counterion and R is Ci-C ₆ alkyl,

[0098] R ₃ taken together with Rn forms a 5- or 6- membered ring that is saturated or unsaturated, and is optionally substituted by one or more Ci-C ₆ alkyl, Ci-C ₆ alkylamine or

Ci-C ₆ alkylsulfonate moieties,

[0100] R ₆ taken together with Re forms a 5- or 6- membered ring that is saturated or unsaturated, and is optionally substituted by one or more Cj-C ₆ alkyl, Ci-C ₆ alkylamine or

Ci-C ₆ alkylsulfonate moieties,

[0101] R ₇ is selected from ~H, -F, -CN, -CO ₂ H, -CO ₂ X, -CO ₂ R, C _r Cio alkyl, Ci-C ₁₀ alkyl that is saturated or unsaturated and is optionally substituted by one or more -F, -Cl, -Br, -

CO ₂ H, -CO ₂ X, -CO ₂ R ₅ -SO ₃ H, -SO ₃ X, -SO ₃ R, where X is a counterion and R is Ci-C ₆ alkyl, or R ₇ is a radical of the formula:

wherein R _{2 , R _!3 , R ₁₄ , R15 and Rj ₆ are each independently selected from -H, -F, -Cl, -Br, -I, - CO ₂ H, -CO ₂ X, -CO ₂ R, -SO3H, -SO ₃ X, and -SO ₃ R, where X is a counterion and R is C ₁ -C ₆ alkyl.

[0102] Exemplary rhodamine dyes useful labels in connection with the present teachings include, but are not limited to, tetramethylrhodamine (TAMRA), 4,7-dichlorotetramethyl rhodamine (DTAMRA), rhodamine X (ROX), 4,7-dichlororhodamine X (DROX), rhodamine 6G (R6G), rhodamine 110 (RIlO), 4,7-dichlororhodamine 110 (RI lO) and the like. Further examples of possible rhodamine dyes that can be used in connection with the present teachings include those described in Menchen, et al. U.S. Patent No. 6,583,168, Bergot, et al. U.S. Patent No. 5,366,860, Lee, et al. U.S. Patent No. 6,191,278, Lam, et al. U.S. Patent No. 6,248,884, Herrmann, et al. U.S. Patent No. 5,750,409, Mao, et al., U.S. Patent No. 6,130,101, Menchen et al. WO 91/03476, Lee, et al. Nucleic Acids Research. 20(10), 2471-2483 (1992) each of which is incorporated herein by reference. [0103] In some cases the designation -1 or -2 is placed after an abbreviation of a particular dye, e.g., TAMRA-I. The "-1" and "-2" designations indicate the particular 5 or 6 dye isomer being used. The 1 and 2 isomers are defined by the elution order (the 1 isomer being the first to elute) of free dye in a reverse-phase chromatographic separation system utilizing a C-8 column and an elution gradient of 15% acetonitrile/85% 0.1 M triethylammonium acetate to 35% acetonitrile / 65% 0.1 M triethylammonium acetate.

[0104] Fluorescein dyes for use in connection with the present teachings can include, for example, any fluorescein dye having the structure:

where Ri-R ₆ are each independently selected from -H, -F, -Cl, -Br, -I, -CN, -CO ₂ H, -CO ₂ X, - CO ₂ R, -SO ₃ H, -SO ₃ X ₅ -SO ₃ R, halogen, Ci-Ci ₀ alkyl, C ₁ -Cj ₀ alkenyl, Ci-Ci ₀ alkynyl, Ci-C ₀ alkoxy, Ci-Ci ₀ alkylamine, Ci-Ci ₀ mercaptyl, Ci-Ci ₀ alkylsulfoiiate, C ₃ -Ci ₀ cycloalkyl, C ₄ - C ₁₀ cycloalkenyl, C ₃ -C ₁₀ heterocyclic, C ₃ -C ₁₀ aromatic, Cs-C ₆ heteroaromatic, where each alkyl, alkenyl, alkynyl, alkoxy, alkylamine, mercaptyl, alkylsulfonate, cycloalkyl, cycloalkenyl, heterocyclic, aromatic and heteroaromatic is optionally further substituted by F, Cl, Br, I, -CO ₂ H, -CO ₂ X, -CO ₂ R, -SO ₃ H, -SO ₃ X or -SO ₃ R where X is a counterion and R is Ci-C ₆ alkyl,

[0105] R ₃ taken together with R ₄ forms a benzo or naphtha ring optionally substituted by - F, -Cl, -Br, -I, -CN, -CO ₂ H, -CO ₂ X, -CO ₂ R, -SO ₃ H, -SO ₃ X, -SO ₃ R, halogen, C]-C ₁₀ alkyl, Cj-Cjo alkenyl, Ci-Cjo alkynyl, C ₁ -C _1O alkoxy, Ci-Ci ₀ alkylamine, Ci-Ci ₀ mercaptyl, Cj-Cto alkylsulfonate, C ₃ -Ci ₀ cycloalkyl, C ₄ -Cj ₀ cycloalkenyl, C ₃ -Ci ₀ heterocyclic, C ₃ -Cj ₀ aromatic, Cs-C ₆ heteroaromatic, where each alkyl, alkenyl, alkynyl, alkoxy, alkylamine, mercaptyl, alkylsulfonate, cycloalkyl, cycloalkenyl, heterocyclic, aromatic and heteroaromatic is optionally further substituted by F, Cl, Br, I, -CO ₂ H, -CO ₂ X, -CO ₂ R, -SO ₃ H, -SO ₃ X or -SO ₃ R where X is a counterion and R is Cj -C ₆ alkyl,

[0106] R ₅ taken together with Rg forms a benzo or naphtha ring optionally substituted by - F, -Cl, -Br, -I, -CN, -CO ₂ H, -CO ₂ X, -CO ₂ R, -SO ₃ H, -SO ₃ X, -SO ₃ R, halogen, C _r Ci ₀ alkyl, Ci-C ₁₀ alkenyl, C ₁ -Ci ₀ alkynyl, Cj-Cio alkoxy, Ci-Ci ₀ alkylamine, Ci-C ₁₀ mercaptyl, Ci-C ₁₀ alkylsulfonate, C ₃ -Ci ₀ cycloalkyl, C ₄ -C ₁₀ cycloalkenyl, C ₃ -Ci ₀ heterocyclic, C ₃ -Ci ₀ aromatic, C ₅ -C ₆ heteroaromatic, where each alkyl, alkenyl, alkynyl, alkoxy, alkylamine, mercaptyl, alkylsulfonate, cycloalkyl, cycloalkenyl, heterocyclic, aromatic and heteroaromatic is optionally further substituted by F, Cl, Br, I, -CO ₂ H, -CO ₂ X, -CO ₂ R, -SO ₃ H, -SO ₃ X or -SO ₃ R where X is a counterion and R is Cj-C ₆ alkyl,

[0107] R ₇ is selected from -H, -F, -CN, -CO ₂ H, -CO ₂ X, -CO ₂ R, Ci-C ₁₀ alkyl, C _r Ci ₀ alkyl that is saturated or unsaturated and is optionally substituted by one or more -F, -Cl, -Br, -

CO ₂ H, -CO ₂ X, -CO ₂ R, -SO ₃ H, -SO ₃ X ₅ -SO ₃ R, where X is a counterion and R is C ₁ -C ₆ alkyl, or R ₇ is a radical of the formula:

wherein R _i2 , Rn, Ri4, R15 and R _i6 are each independently selected from -H, -F, -Cl, -Br, -I, - CO ₂ H, -CO ₂ X, -CO ₂ R, -SO ₃ H, -SO ₃ X, and -SO ₃ R, where X is a counterion and R is C ₁ -C ₆ alkyl.

[0108] Exemplary rhodamine dyes useful labels in connection with the present teachings include, but are not limited to, 6-carboxyfluorescein, 5-carboxyfluorescein, 5-carboxy- 4,7,2',7'-tetrachlorofluorescein, β-carboxy^J^'^'-tetrachloro-fluorescein, 5-carboxy- 4,7,2' ,4 ' ,5 ' ,7 ' -hexachlorofluorescein, 6-carboxy-4,7,2 ' ,4 ' ,5 ' ,T -hexachlorofluorescein, 5- carboxy-4 ' ,5 ' -dichloro-2 ' ,T -dimethoxy-fluorescein, 6-carboxy-4 ' ,5 ' -dichloro-2 ' T -dimeth- oxyfluorescein and S-carboxy^'^'jS'J'-tetrachlorofluorescein.

[0109] In B-L-D, L is a linker whose backbone comprises at least one imidazolium moiety in which the ring nitrogens in each imidazolium moiety are both substituted with non- hydrogen (e.g., a substituent other than hydrogen, such as alkyl) substituents so that the imidazolium ring has a permanent positive charge. For an imidazolium moiety to be considered to be in or part of a linker backbone, a linker backbone must pass through at least two ring atoms of the imidazolium moiety. In other words, the imidazolium ring constitutes a divalent moiety when it is present in a linker backbone. In some embodiments, such substituents can be provided by B, D, or additional linker backbone structure. Alternatively, N-substituents can be non-backbone moieties such as alkyl.

[Ol 10] While at least two imidazolium ring atoms are linked to B, D or additional linker structure, the other ring atoms can be substituted or unsubstituted. For example, when both ring nitrogen atoms are linked directly to B, D or additional linker backbone structure, the three ring carbon atoms (Cl, C4 and C5) can be substituted or unsubstituted. Typically, all three are unsubstituted - in other words, they each have a covalently attached hydrogen atom. Alternatively, one or more non-bridging imidazolium ring atoms can be substituted, e.g., with Ci-Cg substituted or unsubstituted alkyl such as methyl or ethyl.

[0111] These teachings are illustrated, for example, by the resonance structures shown below (Formula I) for an N,N'-disubstituted imidazolium moiety in which each ring nitrogen is covalently linked to additional structure (not shown). As can be seen, the ring contains a net positive charge that is shared by resonance derealization between the two quaternary ring nitrogens.

5 4 5 4

Formula I

{0112] In some embodiments, the imidazolium ring nitrogens are linked covalently to B, D ₅ or additional backbone structure of the linker chain. In Formula I, this is illustrated by the bonds emanating from Nl and N3 whose ends terminate with squiggly lines. Alternatively, an imidazolium moiety may be contained in a linker backbone via one or two of C2, C4 or C5 of the imidazolium moiety, in which case the ring nitrogen is substituted with a non- hydrogen substituent as noted above. For example, Formula II below illustrates embodiments in which an imidazolium moiety has backbone linkages emanating from N3 and from C5. In such embodiments, Nl is substituted with an R group such as C ₁ -C ₆ substituted or unsubstituted allcyl, such as methyl or ethyl, so that the imidazolium moiety is permanently positively charged. More generally, each ring atom of an imidazolium moiety can be hydrogen.

Formula II

[0113] Any of a variety of backbone-imidazolium linker structures can be used. Typically, a linker between B and D will have a linker chain length of from about 4 to about 50 linker chain atoms, or from about 4 to about 30 linker chain atoms, or from about 4 to about 20 linker chain atoms, although shorter and longer linkers may also be used. Several exemplary linker structures are illustrated in the various chemical schemes and the Examples below.

[0114] The junction between a nucleobase B and linker L can be located at any suitable position on the nucleobase. Preferably, the attachment site on the nucleobase is selected so as not to interfere with or eliminate the H-bonding capability of the nucleobase with respect to a complementary nucleobase. When B includes a purine nucleobase, the linker is usually attached to the N-8-position of the purine. When B includes a 7-deazapurine nucleobase, the linker is usually attached to the N-7-position of the 7-deazapurine. When B includes a pyrimidine base, the linkage is attached to the C-5-position of the pyrimidine. In a nucleoside, nucleotide, or polynucleotide sυbunit, a purine or 7-deazapurine is usually attached to a sugar moiety via the N-9-position of the purine or deazapurine, and a pyrimidine is usually attached to a sugar moiety via the N-I -position of the pyrimidine. However, other points of attachment can also be used.

[0115] The particular entity by which a linker is connected to a nucleobase can be any chemical group that is suitable for the purposes of the present invention. A variety of suitable chemical groups are known. For example, the terminal chemical group in the linker that is covalently attached to the nucleobase can be an acetylene moiety (-CsC-), and often is a propargyl moiety (-C≡CCHj-), since such linkage moieties tend to be particularly compatible with a variety of polymerase enzymes used for primer extension. However, non- acetylenic chemical groups are also contemplated. Examples of suitable terminal groups for attachment to a nucleobase can be found in the following exemplary references:

Table 1

[0116] The junction between linker L and dye moiety D can be located at any suitable position on the dye moiety, preferably so that the fluorescent properties of the dye are not adversely affected. For a xanthene-type ring, the linker can be joined to any available carbon atom, or to one of the nitrogen atoms in a rhodamine-type xanthene ring. For a rhodamine dye or fluorescein dye, the substituent positions on the pendent phenyl ring are also available, particularly the positions which are para to C9 of the xanthene ring (5 position), or para to the ortho carboxyl group (6 position), In addition, the particular chemical group by which a linker is connected to a nucleobase can be any chemical group that is suitable for the purposes of the present invention. A variety of chemical groups and points of attachment on various dyes can be found, for example, in US Patents 5,654,442 and 5,188,934 (Menchen et al.), 6,020,481 (Benson et al.), 5,800,996 (Lee et al.), 6,025,505 (Lee et al.), 5,821,356 (Khan et al.), 5,770,716 (Khan et al.), 6,088,379 (Benson et al.), 6,051,719 (Benson et al.), 6,096,875 (Khan et al.), 6,080,868 (Lee et al.), U.S. Patent No. 6,248,884 (Lam et al.), U.S. Patent No. 6,221,604 (Upadhya et al.), U.S. Patent No. 6,465,644 (Menchen et al.), and U.S. Patent No. 6,191,278 (Lee et al.). In some embodiments, for xanthene derivatives that contain a C9 phenyl group, such as a rhodamine dye or fluorescein dye, the linker is attached to the dye via a 5-carboxyρhenyl (para to the xanthene C9 carbon atom) or 6-carboxyphenyl group (meta to the xanthene C9 carbon atom). In some embodiments, for xanthene dyes generally, the linker is preferably attached to a 4-carbon atom or 5-carbon atom on the

xanthene ring. In some embodiments, for rhodamine-type xanthene dyes and rhodamine dyes, the linker is attached to the 3 or 6-nitrogen atom of the xanthene ring. Further guidance for forming conjugates of the invention can be found below with reference to the Examples herein.

[0117] Dye-labeled nucleobase of the invention may also have the form B-L1-D1-L2-D2, wherein B is a nucleobase, Ll and L2 are linkers such that at least one of Ll and L2 comprises an imidazolium-containing linker moiety, and Dl and D2 are members of a fluorescent donor/acceptor pair. In some embodiments, Dl is a donor dye, and D2 is an acceptor dye. In some embodiments, D2 is a donor dye, and Dl is an acceptor dye. For donor/acceptor pairs, it is preferred that the donor dye and acceptor dye have different (non- identical) spectral properties. Thus, although the donor and acceptor may have the same type of aromatic ring structure (e.g., when both the donor and acceptor are fluorescein dyes, or both are rhodamine dyes), different spectral properties can arise for the donor and acceptor due to the nature of the substituents on each one. The donor dye is effective to enhance the intensity of fluorescence emission of the acceptor dye relative to the intensity that would be observed in the absence of the donor dye under the same conditions. Conjugates of this form may be referred to herein as "ET probes", "ET-labeled conjugates" or "ET-labeled nucleotides" because upon excitation of the donor dye, the conjugate can undergo energy transfer (by any of a variety of mechanisms, such as fluorescence resonance energy transfer) from the donor to the acceptor, such that the acceptor dye can then emit fluorescent light at a second wavelength in response thereto.

[0118] The donor dye and acceptor dye can be any fluorescent dye, and are each preferably fluorescent aromatic dyes. For example, the donor and acceptor dye, taken separately, can be a xanthene, rhodamine, dibenzorhodamine, fluorescein, [8,9]benzophenoxazine, cyanine, phthalocyanine, squaraine, or bodipy dye. Furthermore, the donor and acceptor dyes can be linked together using any of a variety of attachment sites on each dye. For example, if Dl is a fluorescein and D2 is a rhodamine (both of which contain pendent phenyl groups attached to C 9 of the xanthene rings), Dl can be linked via its xanthene ring (preferably via C4)) to the pendent phenyl ring of D2 (e.g., via a 5- or 6- carboxy group on the pendent phenyl group). This is referred to as a head to tail arrangement. Alternatively, the positions of the connections can be reversed, such that D2 is linked via its xanthene ring to the pendent phenyl ring of Dl (another example of a head to tail arrangement). In other alternatives, Dl and D2 can be connected tail to tail, via their pendent phenyl rings, or head to head, via their xanthene rings, for example.

[0119] As noted above, at least one of Ll and L2 comprises an iraidazolium-containing linker moiety linker. The properties of such linkers are generally as discussed above for linker L.

[0120] In some embodiments, Ll comprises at least one imidazolium-containing linker moiety and L2 does not. In such embodiments, for L2, any of a variety of linkers can be used to connect Dl to D2. General considerations for forming donor-acceptor conjugates are discussed in US Patents 5,863,727, 5,800,996, 5,945,526, and 6,008,379, for example. In some embodiments, D1-L2-D2 may comprise structure (a), (b) or (c) below:

(a) -D1-R ₂₁ Z,C(O)R ₂₂ R ₂₈ -D2

(b) -Dl-R ₂₈ R ₂₂ C(O)Z ₁ R _2I ^

(c) -DI-R ₂₈ R ₂ 2R2 ₈ -D2

wherein: R ₂ ] is C]-Cs alkyldiyl, Zj is NH, S, or O, R ₂₂ is an alkene, diene, alkyne, or a 5- or 6-membered ring having at least one unsaturated bond or a fused ring structure, and R28 is a bond or spacer group. Details and examples of such inter-dye linkers can be found in U.S. Patent No. 5,800,996, for example. In certain embodiments, R ₂₂ is ethenediyl, ethynediyl, 1,3-butadienediyl, or 1,3-butadiynediyl.

[0121] In some embodiments, L2 comprises at least one imidazolium-containing linker moiety and Ll does not.. In such cases, any of a variety of linkers can be used to connect B to Dl. Descriptions of exemplary linkers can be found in the references in Table 1 above. For example, Ll can be or contain any of the following non-limiting examples:

-CsCCH ₂ NH-

-C^CCH ₂ OCH ₂ CH ₂ NH-

-C ^S CCH ₂ OCH ₂ CH ₂ OCH ₂ CH ₂ NH-

~C=CCH ₂ NHC(O)(CH ₂ ) ₅ NH-

-C=CC(O)NH(CH ₂ ) ₅ NH-

-C=CHC(O)NH(CH ₂ ) ₅ NH-

-C _S C-θ3-C ₆ H ₄ )-(p-C ₆ H ₄ )-C≡C-

-C^C-(P-C ₄ H ₆ )-

-C=C-C=C-

wherein the left-hand ethene or ethyne moiety is linked to the nucleobase, and the right hand bond is typically linked directly to the dye or is linked indirectly to the dye through a carbonyl group.

[0122] When Ll and L2 both comprise at least one imidazolium-containing linker moiety, the structures of Ll and L2 can be the same or different.

[0123] The present disclosure also includes nucleosides and nucleotides containing conjugates in accordance with the invention. Exemplary nucleosides/tides of the present disclosure are illustrate by the following formula:

Formula III

[0124] wherein Wi is OH, H, F, Cl, NH ₂ , N ₃ , or OR, where R is C1-C6 alkyl (e.g., OCH ₃ or OCH ₂ CH ₃ ); W ₂ is OH or a group capable of blocking polymerase-mediated template-directed primer extension (such as H, F, Cl, NH ₂ , N ₃ , or OR, where R is C1-C6 alkyl (e.g., OCH ₃ or OCH ₂ CH ₃ )); W ₃ is OH, or mono-, di- or triphosphate or a phosphate analog thereof; and LB represents a dye-labeled nucleobase conjugate of the present disclosure. For example, LB can comprise a conjugate of the form B-L-D or B-L1-D1-L2-D2, as described herein. In some embodiments, Wi is not OH. In some embodiments, W ₂ is not OH, so that the compound is not

3' extendable. In some embodiments, Wi and W ₂ are each separately selected from H, F, and NH ₂ . In some embodiments, W ₁ is F and W ₂ is H, or W ₁ is H and W ₂ is F, or Wi and W ₂ are each F, or W ₍ and W ₂ are each H. In addition, for each of the foregoing embodiments for Wj alone, W ₂ alone, and Wi and W ₂ in combination, it is contemplated that W ₃ can be OH, monophosphate, diphosphate, or triphosphate. For LB, exemplary nucleobases include adenine, 7-deazaadenine, 7-deaza-8-azaadenine, cytosine, guanine, 7-deazaguanine, 7-deaza-8- azaguanine, thymine, uracil, and inosine.

[0125] For example, in some embodiments, when W ₃ is triphosphate, the present disclosure includes nucleotide triphosphates having the structure shown in the formula below:

Formula IV

[0126] wherein X is H or F. Such terminator nucleotides, and others discussed above which lack a 3 ¹ OH group, find particular application as chain terminating agents in Sanger-type DNA sequencing methods utilizing fluorescent detection, and also in minisequencing. [0127] In some embodiments, the invention includes deoxynucleotide triphosphates having the structure shown in the formula below:

Formula V

[0128] wherein LB is defined as above. Such compounds are examples of 3' extendable nucleotides. Labeled 2'-deoxvnucleotides of this type find particular application as reagents for labeling polymerase extension products, e.g., in the polymerase chain reaction and nick- translation.

[0129] In some embodiments, the invention includes ribonucleotide triphosphates having the structure shown in the formula below:

o- flp P—— oO— 1P— O— i Pf— 0— CH ₂ o.

O- O ^' O-

OH OH _ . „ Formula VI

[0130] wherein LB is defined as above. Labeled nucleotides of this type find particular application as reagents for and in sequencing methods that utilize labile nucleotides having cleavable internucleotide linkages, as discussed for example in US Patent 5,939,292 (Gelfand et al.), Eckstein, Nucl. Acids Res. 16:9947-9959 (1988), U.S. Patent 6,887,690 (Fisher et al.), and Shaw, Nucl Acids Res. 23:4495 (1995).

[0131] The various compounds of the present disclosure (e.g., nucleobase-linker-dye conjugates, nucleosides, and nucleotides) may be prepared by any suitable synthetic method. Typically, conjugates are formed using a modular approach in which a nucleobase (which optionally may be provided in the form of a nucleoside or nucleotide .containing the nucleobase, for example), a first dye, a second dye (if present), and one or more linkers or linker precursors, are combined in serial and/or parallel steps to produce the desired labeled product. Several exemplary approaches are illustrated in the Examples below, which describe syntheses of linkers and conjugates containing linkers of various lengths and compositions.

[0132] In some embodiments the label can optionally be attached to a linker through a linkage formed by the reaction of a nucleophilic moiety of the linker with a complementary functionality located on the label. The complementary functionality can be, for example, isothiocyanate, isocyanate, acyl azide, N-hydroxysuccinimide (NHS) ester, sulfonyl chloride, aldehyde or glyoxal, epoxide, carbonate, aryl halide, imidoester, carbodiimide, 4,6- dichlorotriazinylamine, carboxylic acid anhydride, or other active carboxylate, see Hermanson, Bioconiugate Techniques. Academic Press, 1996. For example, in some embodiments the complementary functionality can optionally be an activated NHS ester that reacts with a nucleophilic moiety on the linker. The activated NHS ester on the label can be formed by reacting a label, such as a carboxylate-complementary functionality, with dicyclohexylcarbodiimide and N-hydroxysuccinimide to form the NHS ester. By way of example, Table 1 shows a sampling of representative complementary functionalities and resulting linkages formed by reaction of the complementary functionality with an amine moiety on the linker.

TABLE 1

[0133] When compounds of the present teachings include a detectable label, the label can be any moiety that, when attached to the compounds of the present teachings, renders the compound to which the label is attached detectable using known detection means. Examples of such labels include but are not limited to fluorophores, chromophores, radioisotopes, spin- labels, enzyme labels, and chemiluminescent labels. Furthermore, the label can optionally be, for example, a ligand, such as an antigen, or biotin, which can bind specifically with high affinity to a detectable anti-ligand, such as a labeled antibody or avidin, In some embodiments, detectable labels comprise fluorescent dyes such as fluorescein, rhodamine, rhodol or energy transfer dyes. For example, various fluorescent dyes are described in U.S. Patent Application Publication US 2002/0102590 Al, which is incorporated herein by reference.

[0134] Examples 1 to 4 (Scheme 1) illustrate methods for forming linker synthons containing one, three, or four backbone imidazolium moieties.

[0135] Example 5 provides an alternative protocol to that of Example 2 in which 1,3- diiodopropane is used instead of 1,3-dibromopropane to alkylate protected aminoethyl imidazole A3, for further reaction with additional imidazole-containing synthons such as compound A6.

[0136] Examples 6 to 8 (Scheme 2) illustrate methods for forming linker synthons containing one, three, or four backbone imidazolium moieties using protection schemes that are different from those in Examples 1 to 4, and a convergent synthesis for forming a linker synthon containing six backbone imidazolium moieties.

[0137] Examples 9 to 12 (Schemes 3 and 4) illustrate methods for forming linker synthons containing one, three, or four backbone imidazolium moieties in which two imidazolium moieties are separated by an ethylene bridge instead of the propylene bridges shown in

Schemes 1 and 2.

[0138] Examples 13 to 15 (Scheme 5) illustrate methods for forming linker synthons containing one or two backbone imidazolium moieties using another protection scheme.

[0139] Examples 16 to 18C (Scheme 6) illustrate model reactions for conjugating an imidazolium linker moiety to an amino compound (represented by 3,4- dimethoxyphenethylamine) and a fluorescent dye label (FlO).

[0140] Examples 19 to 22 (Schemes 7 and 8) a modular, stepwise protocol for forming a hexaimidazolium linker labeled with an energy transfer dye.

[0141] Example 23 (Scheme 9) illustrates synthesis of a dye-labeled nucleoside triphosphate comprising a hexameric backbone-irm'dazolium moiety.

[0142] Example 24 (Scheme 10) illustrates synthesis of a dye-labeled nucleoside triphosphate comprising one backbone-imidazolium, built stepwise from a nucleoside triphosphate containing an aminoethoxypropargyl moiety by adding an imidazolium- containing synthon and then a fluorescent label. This contrasts with the approach shown in

Schemes 7 to 9 in which the nucleoside triphosphate is added in the last step.

[0143] Schemes 11 and 12 illustrate syntheses of dye-labeled nucleoside triphosphate comprising two and three backbone-imidazolium moieties, respectively, built stepwise from a nucleoside triphosphate.

[0144] The present disclosure also provides polynucleotides and mixtures of polynucleotides that contain one or more different nucleobase-linker-dye conjugates of the type discussed above.

Such polynucleotides are useful in a number of important contexts, such as DNA sequencing, ligation assays, the polymerase chain reaction (PCR), probe hybridization assays, and various other sequence detection or quantitation methods.

[0145] Polynucleotide(s) may be formed by any appropriate method. For example, polynucleotides containing nucleobase linker dye conjugates may be synthesized enzymatically, e.g., using a DNA or RNA polymerase, nucleotidyl transferase, ligase, or other enzymes, e.g., Stryer, Biochemistry, Chapter 24, W.H. Freeman and Company (1981), or by chemical synthesis, e.g., by the phosphoramidite method, the phosphite-triester method, or the like. Labels may be introduced during enzymatic synthesis utilizing labeled nucleotide triphosphate monomers as described above, or during chemical synthesis using labeled non-nucleoside or nucleoside phosphoramidites, or may be introduced subsequent to synthesis. Exemplary methods for forming labeled polynucleotides can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, NY (1989), US Patents 6,008,379 (Benson et al.), and references cited therein, for example. [0146] For example, if a labeled polynucleotide is made using enzymatic synthesis, the following procedure may be used. An oligonucleotide primer is annealed to a complementary sequence in a template DNA strand. A mixture of deoxynucleotide triphosphates (such as dGTP, dATP, dCTP, and dTTP) is added, where at least one of the deoxynucleotides contains a nucleobase-dye conjugate of the invention. In the presence of a polymerase enzyme, a dye- labeled polynucleotide is formed by incorporation of a labeled deoxynucleotide during polymerase-mediated strand synthesis. In an alternative enzymatic synthesis method, two primers are used instead of one, one primer complementary to the + strand and the other complementary to the - strand of the target, the polymerase is a thermostable polymerase, and the reaction temperature is cycled between a denaturation temperature and an extension temperature, thereby exponentially synthesizing (amplifying) a labeled complement to the target sequence by PCR, e.g., PCR Protocols, Innis et al. eds., Academic Press (1990). [0147] In some embodiments of primer extension, the primer extension reagent includes a thermostable polymerase. Examples of thermostable polymerases include but are not limited to rTth DNA polymerase, Bst DNA polymerase, Vent DNA polymerase, Pfu DNA polymerase, or Tag polymerase enzyme as described in, for example, PCR Primer: A Laboratory Manual, Dieffenbach and Dveksler, eds., CSHL Press (1995). In some embodiments, the thermostable polymerase can be Tag DNA polymerase, or a mutant Tag polymerase enzyme having, for example, a mutation at the F667 position as described in, for example, Tabor and Richardson, EP 0 655506. In some embodiments, the mutation at the F667 position can be F667Y. In an additional embodiment of the primer extension reaction of the present teachings, Tag polymerase enzyme can be a mutant that includes, in addition to the F667Y mutation, one or more mutations at the 660, 664, 665 and/or the 681 positions.

See U.S. Patent 6,265,193. In some embodiments, representative mutations at the 660, 664, 665 and/or the 681 positions include, but are not limited to, R660D, R660E, R660C, R660S, R660P, and E681G. In some embodiments, the mutant Tag polymerase enzyme includes at least one of the mutations R660C or R660S, R660P and F667Y.

[0148] Labeled polynucleotides may be chemically synthesized using any suitable method, such as the phosphoramidite method Detailed descriptions of the chemistry used to form polynucleotides by the phosphoramidite method are provided elsewhere, e.g., Caruthers et al., U.S. Patents No. 4,458,066 and 4,415,732, Caruthers et al., Genetic Engineering 4: 1-17 (1982), Users Manual Model 392 and 394 DNA/RNA Synthesizers, pages 6-1 through 6-22, Applied Biosystems, Part No. 901237 (1991). Descriptions of the phosphoramidite method and other synthesis methods for making polynucleotides containing standard phosphodiester linkages or linkage analogs can be found in Gait, Oligonucleotide Synthesis, IRL Press (1990), and S. Agrawal, Protocols for Oligonucleotides and Analogs, Methods in Molecular Biology Vol. 20, Humana Press, Totowa, NJ (1993).

[0149] The phosphoramidite method is a preferred chemical method because of its efficient and rapid coupling and the stability of the starting materials. The synthesis is performed with a growing polynucleotide chain attached to a solid support, so that excess reagents, which are in the liquid phase, can be easily removed by filtration, thereby eliminating the need for purification steps between synthesis cycles.

[0150] Nucleobase-dye conjugates of the present disclosure are suited for any method utilizing fluorescent detection, particularly methods requiring simultaneous detection of analytes which are not well separated by electrophoresis. Aspects of the present disclosure are particularly well suited for detecting classes of polynucleotides that have been subjected to a biochemical separation procedure, such as electrophoresis.

[0151] In some embodiments, the present disclosure provides methods of identifying one or more polynucleotide(s). The method utilizes one or more labeled different-sequence polynucleotides, which may have the same lengths or different lengths, wherein each different-sequence polynucleotide contains a unique nucleobase-dye conjugate. The one or more labeled different-sequence polynucleotides are separated by electrophoresis to separate different-sequence polynucleotides on the basis of size. Each different-sequence polynucleotide can then be identified on the basis of its electrophoretic mobility and, optionally, fluorescence signal.

[0152] In some embodiments, each different polynucleotide is identifiable on the basis of a unique combination of electrophoretic mobility and fluorescence signal. For example, two

different polynucleotides may contain identical dye moieties but may exhibit different electrophoretic mobilities. Alternatively, two different polynucleotides can contain different dye moieties that produce distinct (spectrally resolvable) fluorescence signals but can exhibit the same electrophoretic mobilities. In another example, different polynucleotides can differ in both their fluorescence signals and mobilities.

[0153] In some embodiments, the method can be used in a multiplex format in which different labeled polynucleotides are formed by reaction with (i) a plurality of different target sequences and (ii) a plurality of different polynucleotides that are complementary to the target sequences. For example, the different polynucleotide can be designed to undergo a change in structure after hybridization to their complementary target sequences in a polynucleotide sample, e.g., due to modification by enzyme action, thereby producing different labeled polynucleotides having unique combinations of mobility and fluorescence to allow identification. Such reactions can be performed simultaneously in a single reaction mixture or can be performed in separate reaction mixtures that can be combined prior to electrophoretic separation. Several exemplary assay formats for producing such labeled polynucleotides are discussed below.

[0154] Sanger-type sequencing involves the synthesis of a DNA strand by a DNA polymerase in vitro using a single-stranded or double-stranded DNA template whose sequence is to be determined or confirmed. Synthesis is initiated at a defined site based on where an oligonucleotide primer anneals to the template. The synthesis reaction is terminated by incorporation of a nucleotide analog that will not support continued DNA elongation. Exemplary chain-terminating nucleotide analogs include the 2',3'-dideoxynucleoside 5'- triphosphates (ddNTPs) which lack the 3'-OH group necessary for 5' to 3' DNA chain elongation. When proper proportions of dNTPs (2'-deoxynucleoside 5 -triphosphates) and one of the four ddNTPs are used, enzyme-catalyzed polymerization will be terminated in a fraction of the population of chains at each site where the ddNTP is incorporated. If labeled ddNTPs are used for each reaction, a desired sequence read can be obtained by detection of the fluorescence signals of the terminated chains during or after separation by high-resolution electrophoresis. In the chain termination method, dyes of the invention can be attached to either sequencing primers or terminator nucleotides.

[0155] In "fragment analysis" or "genetic analysis" methods, labeled polynucleotide fragments can be generated through template-directed enzymatic synthesis using labeled primers or nucleotides, e.g., by polynucleotide ligation or polymerase-directed primer extension. The resultant fragments are then subjected to a size-dependent separation process, e.g.,

31 SUBSTTTUTE SHEET (RULE 26)

electrophoresis or chromatography, and the separated fragments are detected, e.g., by laser- induced fluorescence. In a particular embodiment, multiple classes of polynucleotides are separated simultaneously and the different classes are distinguished by spectrally resolvable labels.

[0156] A fragment analysis method, known as amplified fragment length polymorphism detection (AmpFLP), is based on amplified fragment length polymorphisms, i.e., restriction fragment length polymorphisms that are amplified by PCR. These amplified fragments of varying size serve as linked markers for following mutant genes in family lineages. The closer the amplified fragment is to the mutant gene on the chromosome, the higher the linkage correlation. Because genes for many genetic disorders have not been identified, these linkage markers serve to help evaluate disease risk or paternity. In the AmpFLP technique, the polynucleotides may be labeled by using a labeled polynucleotide PCR primer, or by utilizing labeled nucleotide triphosphates in the PCR.

[0157] m another fragment analysis method, known as nick translation, one or more unlabeled nucleotide subunits in a double-stranded DNA molecule are replaced with labeled subunits. Free 3'-hydroxyl groups are created within the unlabeled DNA by "nicks" caused by treatment with deoxyribonuclease I (DNAase I). The DNA polymerase I then catalyzes the addition of one or more labeled nucleotides to the 3 -hydroxyl of the nick. At the same time, the 5' to 3'-exonuclease activity of this enzyme can remove one or more nucleotide subunits from the 5'-phosphoryl terminus of the nick. A new nucleotide with a free 3'-OH group is incorporated at the position of the excised nucleotide, and the nick is shifted along by one nucleotide unit in the 3' direction. This 3' shift will result in the sequential addition of new labeled nucleotides to the DNA with the removal of existing unlabeled nucleotides. The nick- translated polynucleotide is then analyzed using a separation process, e.g., electrophoresis. [0158] Another exemplary fragment analysis method is based on the variable number of tandem repeats, or VNTRs. VNTRs are regions of double-stranded DNA that contain adjacent multiple copies of a particular sequence, with the number of repeating units being variable among different members of a population (e.g., of humans). Examples of VNTR loci are pYNZ22, pMCT118, and Apo B. A subset of VNTR methods are based on the detection of microsatellite repeats, or short tandem repeats (STRs), i.e., tandem repeats of DNA characterized by a short (2-4 bases) repeated sequence. One of the most abundant interspersed repetitive DNA families in humans is the (dC-dA)n~(dG-dT)n dinucleotide repeat family (also called the (CA)n dinucleotide repeat family). There are thought to be as many as 50,000 to 100,000 (CA)n repeat regions in the human genome, typically with 15-30 repeats per block.

Many of these repeat regions are polymorphic in length and can therefore serve as useful genetic markers. Preferably, in VNTR or STR methods, label is introduced into the polynucleotide fragments by using a dye-labeled PCR primer.

[0159] In another example, known sometimes as an oligonucleotide ligation assay (OLA), two polynucleotides (probe pair) which are complementary to adjacent regions in a target sequence are hybridized to the target region of a polynucleotide, to create a nicked duplex structure in which the ends of the two polynucleotide abut each other. When the ends of the hybridized polynucleotide probes match (basepair with) corresponding subunits in the target, the two probes can be joined by ligation, e.g., by treatment with ligase. The ligated product is then detected, evidencing the presence of the target sequence. In a modification of this approach, known as the ligation chain reaction (or ligation amplification reaction), the ligation product acts as a template for a second pair of polynucleotide probes which are complementary to the ligated product from the first pair. With continued cycles of denaturation, reannealing and ligation in the presence of the two complementary pairs of probe, the target sequence is amplified exponentially, allowing very small amounts of target sequence to be detected and/or amplified. Exemplary conditions for carrying out such processes, including chemical ligation formats, are described in US Patents 5,962,223 (Whiteley et al.), 4,988,617 (Landegren et al.), and 5,476,930 (Letsinger et al,), and European Patent Publications EP 246864A (Carr et al.), EP 336731A (Wallace), and EP 324616A (Royer et al.).

[0160] Conveniently, a fragment analysis method such as any of those discussed above can be performed in a multi-probe format, in which a sample is reacted with a plurality of different polynucleotide probes or probe sets which are each specific for a different target sequence, such as different alleles of a genetic locus and/or different loci. The probes are designed to have a unique combination of mobility and fluorescence signal, to permit specific detection of the individual probes or probe products that are generated in the assay as a result of the presence of the different target sequences.

[0161] In some embodiments of the present disclosure, polynucleotides may be subjected to a size-dependent separation process. Without being limiting in any way, the size- dependent separation process can comprise electrophoresis, chromatography, or hybridization to a set of polynucleotide probes that bind to the fragments in a sequence-dependent manner as described in, for example, Drmanac et al., Nature Biotechnology, 16: 54-58 (1998), Ramsay, Nature Biotechnology, 16: 40-44 (1998) and U.S. Patent No. 5,202,231. In some embodiments, subsequent to separation or hybridization, the polynucleotides are detected, by,

for example, laser-induced fluorescence. Further, in some embodiments, multiple classes of polynucleotides can be separated or hybridized simultaneously and the different classes can be distinguished by a set of spectrally resolvable labels.

[0162] Methods for electrophoresis of nucleic acids are well known and are described, for example in Rickwood and Hames, Eds., Gel Electrophoresis of Nucleic Acids: A Practical Approach, IRL Press Limited, London (1981), Osterman, Methods of Protein and Nucleic Acid Research, Vol. 1 Springer-Verlag, Berlin (1984), Sambrook et al. (1989, supra), P.D. Grossman and J.C. Colburn, Capillary Electrophoresis: Theory and Practice, Academic Press, Inc., NY (1992), and U.S. Patents 5,374,527, 5,624,800 and/or 5,552,028. Typically, the electrophoretic matrix contains crosslinked or uncrosslinked polyacrylamide having a concentration (weight to volume) of between about 2-20 weight percent, and often about 4 to 8 percent. For DNA sequencing, the electrophoresis matrix usually includes a denaturing agent such as urea, formamide, or the like. Detailed exemplary procedures for forming such matrices are given by Maniatis et al., "Fractionation of Low Molecular Weight DNA and RNA in Polyacrylamide Gels Containing 98% Formamide or 7 M Urea," in Methods in Enzymology, 65: 299-305 (1980), Sambrook et al. (1989, supra), and ABI PRISM™ 377 DNA Sequencer User's Manual, Rev. A, January 1995, Chapter 2 (p/n 903433), Applied Biosystems, Foster City, CA) and in U.S. Patent 6,355,709 (Madabhushi et al.), U.S. Patent 6,706,162 (Voss et al.), WO 03/008074 (Hacker et al.), WO 04/011513 (Lau), and WO 04/104054 (Lau). A variety of suitable electrophoresis media are also commercially available from Applied Biosystems ("POP" polymers) and other vendors, including non-crosslinked media, for use with automated instruments such as the Applied Biosystems "3700", "3100", and "3130" Instruments, by way of example. [0163] Optimal electrophoresis conditions, e.g., polymer concentration, pH, temperature, voltage, concentration of denaturing agent, employed in a particular separation depends on many factors, including the size range of the nucleic acids to be separated, their base compositions, whether they are single stranded or double stranded, and the nature of the polynucleotides for which information is sought by electrophoresis. Accordingly application of the invention may require preliminary testing to optimize conditions for particular separations. [0164] During or after separation, labeled polynucleotides can be detected or identified by recording fluorescence signals (or other detectable signals) and migration times (or migration distances) of the separated polynucleotides, or by constructing a chart of relative fluorescent and order of migration of the polynucleotides (e.g., as an electropherogram). For example, to perform fluorescence detection, the labeled polynucleotides can be illuminated by standard means, e.g. a high intensity mercury vapor lamp, a laser, or the like. Typically, the labeled

polynucleotides are illuminated by laser light generated by a He-Ne gas laser or a solid-state diode laser. The fluorescence signals can then be detected by a light-sensitive detector, e.g., a photomultiplier tube, a charged-coupled device, or the like. Exemplary electrophoresis detection systems are described elsewhere, e.g., U.S. Patent Nos. 5,543,026, 5,274,240, 4,879,012, 5,091,652 and 4,811,218.

[0165] In some embodiments, labeled nucleoside triphosphates that comprise backbone- imazolium-containing linker moieties are designed to have a net neutral or net positive charge under selected conditions. In some embodiments, such nucleotide triphosphates have a net neutral or net positive charge at a pH of 7 or greater, or a pH of 8 or greater. For example, the triphosphate portion of a nucleoside triphosphate typically bears 3 to 4 negative charges, depending on the pH of the environment (3 negative charges if the pH is below about 6, and 4 negative charges if the pH is above about 6). An simple fluorescein dye backbone (without additional charged substituents) typically contains about 2 negative charges under neutral or basic pH conditions. Accordingly, a linker that comprises at least 6 positive charges (e.g., containing at least 6 backbone-imidazolium moieties) should suffice to confer an overall net neutral or positive charge to the nucleotide triphosphate. If a simple rhodamine dye is present instead (typically having a net neutral charge), then a linker moiety containing at least 4 backbone-imidazolium moieties should be sufficient to confer an overall net neutral or positive charge to the nucleotide triphosphate. Linkers containing fewer backbone-irnidazolium moieties can be used if additional positive charge are present elsewhere in the labeled nucleotide triphosphate.

[0166] In some embodiments, nucleoside triphosphates that comprise backbone-imidazolium moieties and that have a net neutral or net positive charge can be used advantageously in primer extension or other reactions that are subsequently subjected to electrophoresis separation since such nucleoside triphosphates and their breakdown products will migrate away from, or only very slowly toward, the anode so that overlap with polynucleotides of interest can be reduced or avoided. In some embodiments, primer extension reaction mixtures are loaded into a capillary or other electrophoretic pathway without prior removal of residual triphosphates. In such cases, it is typically desirable to reduce the ionic strength of the reaction mixture by dilution into water or other aqueous solution having a very low or zero ionic strength prior to loading the mixture into the capillary. Alternatively, the mixture can be first treated with ethanol precipitation, size exclusion chromatography, or other method to remove residual unincorporated nucleoside triphosphates and other small molecules prior to performing electrophoresis.

[0167] Also provided are kits for performing the various methods of the invention. For nucleic acid sequencing, the kit comprises at least one labeled nucleoside triphosphate comprising a conjugate described herein. The kit may also include one or more of the following components: a 3 '-extendable primer, a polymerase enzyme, one or more 3' extendable nucleotides which are not labeled with conjugate, and/or a buffering agent. In some embodiments, the kit includes at least one labeled nucleoside triphosphate that is nonextendable. In other embodiments, the kit comprises four different labeled nucleoside triphosphates which are complementary to A, C, T and G, and each of which contains a distinct conjugate as described herein. In some embodiments, the labeled nucleoside triphosphates are nonextendable. In some embodiments, the labeled nucleoside triphosphates are extendable ribonucleoside triphosphates. In some embodiments, the kit comprises at least one labeled, nonextendable nucleoside triphosphate comprising a conjugate described herein, and one or more of the following components: a 3 '-extendable primer, a polymerase enzyme, and/or a buffering agent.

[0168] The present teachings can be further understood in light of the following non- limiting examples.

EXAMPLES Materials and Methods

Unless indicated otherwise, all reagents and anhydrous solvents were purchased from Aldrich Chemicals. Thin layer chromatography (TLC) analysis was conducted on aluminum plates precoated with 250 μm layers of silica gel 60-F254. Compounds were located by UV- VIS lamp and/or by charring with aqueous KMnO ₄ or ninhydrin/butanol solution. Flash column chromatography purification was carried out using EM Science silica gel 60 angstrom (230-400 Mesh ASTM). NMR spectra were recorded in deuterated solvents (CDCl ₃ , CH ₃ OD, DMSO-d6 and D ₂ O with an internal Me ₄ Si standard, δ 0). ¹ H NMR spectra were recorded at 400 MHz.

Example 1 fOO5O138-O83.11

N-2-Tert-Butoxycarbonylaminoethyl Imidazole

To a stirring solution of 5% (aq) sodium carbonate (25 g, 238 mmol) was added 22.8 g (100 mmol) N-2-aminoethyl imidazole AJ. (prepared in accordance with Alvarez-Builla et al., Synthetic Communications 21(4):535-544, 1991), followed by 175 mL THF (tetra- hydrofuran) and a first portion of di-terr-butyl dicarbonate A2 (25.6 g, 117 mmol). The mixture was stirred at room temperature for 3 h, during which it warmed, cooled, became clear, then cloudy. A second portion of A2 (6 g) was then added and stirred over night at room temperature. The mixture was then diluted to 1000 mL with ethyl acetate (EA). After the solvent layers separated, the aqueous layer was extracted twice with 200 mL of ethyl acetate, and finally with 150 mL of ethyl acetate. The combined organic portions were washed twice with 150 mL portions of brine, dried over Na ₂ SO ₄ , filtered, and evaporated under reduced pressure to yield 15 g (71 mmole) of A3 as an oil.

Example 2

[0050138-087.11

N ¹ -3-Bromopropyl-N ³ -2-Tert-Butoxycarbonylaminoethyl Imidazolium Bromide

4.22 g (20 mmol) of N-2-tert-butoxycarbonylaminoethyl imidazole A3 and 20 mL (40 g, 200 mmol) 1,3-dibromopropane A4 were dissolved in 20 mL DMF (dimethylformamide) in a 100 mL flask and heated overnight at 8O ⁰ C using an oil bath. When the reaction was complete based on NMR analysis of an aliquot, the DMF was removed by high vacuum

rotoevaporation. The concentrated material was chromatographed on a 130 mm x 75 mm bed of silica gel 60 (Merck) packed in 20:1 dichloromethane:methanol (DCM:M) eluted with (1) 600 mL of 20:1 DCM:M, (2) 1 L of 10:1 DCM:M, then (3) 2 L of 5:1 DCM:M, during which fractions of 225 mL were collected. TLC analyses of fractions were performed on silica eluted with 5:1 DCM:M, and product was visualized with iodine and ninhydrin. Product- containing fractions were collected and dried under reduced pressure, yielding 6.8 g (16.5 mmol) A5 as an opaque thick oil.

Example 3 t-Boc-Protected Linker Element Containing Three Imidazolium Moieties 3.3 g (8 mmol) of A5 and 10.7 g (60 mmol) A6 were dissolved in 30 mL DMF in a 100 mL flask and heated at 85°C using an oil bath. After NMR analysis of an aliquot indicated completion of the reaction (after about 16 h of heating), the reaction mixture was filtered through a coarse frit (which removed symmetrical side-product AT) and solvent was removed under high vacuum. The resultant product was dispersed in 35 mL of DCM, poured into 140 mL of THF, and stirred over night, yielding a gummy precipitate in a clear supernatant. After decantation, the precipitate was re-precipitated in the same manner (35 mL DCM/140 mL THF) twice more, yielding 4.14 g (7 mmol) product A8 as a yellow solid in a ratio of about 20: 1 relative to starting material A6 based on NMR analysis.

Example 4

[0050138-095.1] t-Boc-Protected Bromide Linker Element Containing Three Imidazolium Moieties t-Boc-protected tri-imidazolium A8 (960 mg, 1.63 mrnol) was dissolved in 10 mL warm DMF. A salt precipitate was removed using a 20 mL disposable polypropylene syringe with PTF6 syringe filter. 1,3-Dibromopropane A4 (6.7 g, 33 mmol) was then added and the mixture was heated at 8O ⁰ C over night, followed by high vacuum removal of solvent. The product was resuspended in 20 mL DMF, filtered through a medium frit, followed by washing the frit with 3 mL of DMF. The collected solution was subjected to reduced pressure to remove solvent, yielding 1 g of white solid. The white solid was suspended and sonicated in acetonitrile. Solid product was recovered by suction filtration on a medium frit and vacuum dried, yielding 700 mg (0.88 mmol) of A9 white solid.

Example 5

N ¹ -3-Iodopropyl-N ³ -2-Tert-Butoxycarbonylaminoethyl Imidazolium Iodide This example describes a reaction similar to that of Example 2, except that 1,3- diiodopropane was used in place of 1,3-dibromopropane.

A3 (2.2 g, 10 mmol, see Example 1 for synthesis) and 1,3-diiodopropane AlO (H g, 37.3 mmol) were dissolved in 25 mL THF in a 50 ml flask equipped with a reflux condenser, and the mixture was refluxed in an oil bath at 65 ⁰ C over night, forming a large precipitate. After refluxing, the flask was removed from the oil bath and allowed to cool to room temperature. The solid was removed by filtration on a medium frit, chased with 5 mL THF, and then dried under high vacuum, yielding 1.2 g of solid that was identified as the adduct of 1,3-diiodopropane AlO with two molecules of A3. (This adduct, All, was not used further in this example.) The THF filtrate was chromatographed on a 50 by 50 mm column of silica with 5:1 DCM/M. Product-containing fractions were combined, rotoevaporated and then rechromatographed on a 50 by 50 mm silica column as above with 10:1 DCM:M and then 5:1 DCM:M. Except for an early fraction that contained a substantial amount of residual AlO, product-containing fractions were pooled and evaporated, yielding 2.8 g (5.5 mmol) of gummy oil that solidified into a soft yellow solid.

Example 6

N^-Bromopropyl-N^-Carboxypropyl Imidazole Ethyl Ester 4.4 g (24.2 mmol) of N-3-carboxypropyl imidazole ethyl ester Bl and 13 mL (24.4 g, 121 mmol) 1,3-dibromopropane (A4) were dissolved in 25 mL DMF in a 100 mL flask and heated over night at 80 ⁰ C. The DMF was removed under reduced pressure, yielding 10 g of a yellow oil. The oil was chromatographed on a 75 mm by 150 mm bed of silica gel 60 packed in 20:1 DCM:M, that was eluted with (I) I L of 20:1 DCM:M, (2) 1 L 10:1 DCM:M, then (3) 3 L of 4:1 DCM:M (225 mL fractions). Thin layer chromatography (TLC) analyses of fractions were performed on silica eluted with 5:1 DCM:M, and product was visualized with iodine. Product-containing fractions were collected and concentrated under reduced pressure, yielding 7.6 g (19.8 mmol) B2 as an opaque oil.

Example 7 r0050138-097.11

Ethyl Ester of Linker Element Containing Three Imidazolium Moieties Compounds B2 (3.6 g) and A6 (14 g, prepared in accordance with Diez-Barra, E., et. al., Heterocvcles 34(7):1365-1373, 1992) were dissolved in DMF (15 mL) in a 250 mL flask, and the reaction was heated at 8O ⁰ C over night. Solvent was then removed under reduced pressure, and the resultant product was dispersed in 40 mL DCM followed by addition of 120 mL of THF. After the mixture was stirred at room temperature for one hour, product was collected by suction filtration on # 50 Whatman filter paper. The resultant pasty solid, which melts very quickly in ambient air, was transferred to a 250 mL Erlenmeyer flask and dispersed again in 40 mL of DCM, followed by addition of 120 mL THF. After the mixture was stirred for 1 h, product was collected on # 50 Whatman filter paper, transferred to a flask, and subjected to high vacuum, yielding 4.5 g of solid. NMR analysis indicated a ratio of product compound B4 to compound A6 of about 33:1 (based on integrating the imidazole protons at 7.7 ppm), and a ratio of desired product B4 to symmetrical diester B3 of about 14 (based on integrating the imidazolium protons at 9.4, 9.5 and 9.6).

Example 8 IO050138-099.1

Linker Element Containing Six Imidazolium Moieties

6SO mg of A9 (860 μmol, Example 4) was dissolved in 8 mL DMF by heating in an 8O ⁰ C oil bath. 500 mg (890 μmol) of solid B4 was added, which dissolved slowly with heating in the oil bath followed by formation of a precipitate. After a total of about 15 h of heating in the oil bath, the resultant solid was collected slowly on a 45 mm diameter #50 Whatman filter and then subjected to high vacuum over night, yielding 830 mg of doubly protected CJ ₁ .

Product Cl was dissolved in 5 mL cone HCl (12 M) and refluxed for 1 h. The mixture started bubbling as soon as the HCl was added. Solvent was then removed under reduced pressure, yielding a thick oil. Ten mL water was added and then removed under reduced pressure, yielding a thick paste. High vacuum over night yielded 850 mg (850 μmol) of solid C2. NMR analysis in D ₂ O (with slight amount of K ₂ CO ₃ to aid dissolution) indicated that the t-butyl group and ethyl ester had been hydrolyzed.

Example 9 r0050138-033.11

N-trimethylsilylimidazole Dl (2.8 g, 20 mmol) and ethyl 4-bromobutanoate D2 (4 g, 20 mmol) were dissolved in DMF (8 mL) in a 25 mL flask and heated over night in a 7O ⁰ C oil bath. The reaction was poured into 100 mL of water and was saturated with solid sodium bicarbonate. The resulting liquid was decanted into a separatory tunnel, chased with 10 mL of water, and extracted three times with 100 mL portions of DCM. Analysis of each DCM extract by thin layer chromatography (silica, 5:1 DCM/M visualized with iodine) indicated that all product was extracted by the second extract. The first and second DCM extracts were pooled, washed with water and then by brine, dried with Na ₂ SO ₄ , filtered, and evaporated to form an oil. The oil was chromatographed on silica (50 by 180 mm, 20:1 DCM/M). Product fractions were pooled and rotoevaporated followed by high vacuum to remove residual solvent, yielding 1.3 g (7.1 mmol) of D3 as a light yellow oil.

Example 10 [0050138-049.11

D3 (1.82 g, 10 mmol) and A4 (10 g, 50 mmol) were mixed in DMF (10 mL) in a 50 mL flask and heated at 7O ⁰ C for 16 h. The DMF was removed by rotoevaporation under high vacuum. The product was chromatographed on silica (50 by 150 mm with 10:1 DCM/M). Product-containing fractions were analyzed by TLC (silica developed with 5:1 DCM:M), visualized with iodine, pooled and rotoevaporated, yielding 2.88 g (7.5 mmol) D4 of a cloudy oil.

Example 11

[0040314-153.1]

In a 250 mL 3-neck flask with mechanical stirring and reflux condenser with argon atmosphere and bubbler, imidazole El (27.25g, 400 mmol), (CH ₃ (CH ₂ ) ₃ ] ₄ N)HSθ4 (3.86g, 1 1.4 mmol), and KOH (26.8g, 480 mmol) were added with stirring, forming a thick liquid. After 45 min, 1,2-dichloroethane E2 (19.8 g, 15.2 mL, 200 mmol, 1 equiv) was added in one portion and heated gently with a heating mantle, during which gas was evolved and the mixture became warm. The heating mantle was then removed and the reaction was stirred overnight. The reaction was then diluted with 300 mL of ethanol and filtered on #2 filter paper on a Buchner funnel. The solid was washed with 20 mL ethanol. The solid was dried under reduced pressure, dissolved in about 200 ml of 8:1 DCM/M, and chromatographed on a silica column, 75 by 180 mm, packed with 10:1 DCM/M. Elution was performed with 10:1 DCM/M. Fractions were analyzed by TLC with 5:1 DCM/M and visualized using iodine. Fractions that contained 1,2-diimidazole E3 were combined, dried, and rechromatographed

through a 75 by 180 mm silica column packed with 10:1 DCM/M. Elution was performed initially with 10:1 DCM/M until TLC analysis of fractions showed that imidazole El stopped eluting, then with 5:1 DCM/M until E3 stopped eluting. AU fractions contained E3 were combined and dried, yielding 4.4 g (27 mmol) of solid E3.

Example 12

[0050138-053.1]

D4 (2.88g, 7.52 mmol) and E3 (609mg, 3.76 mmol, 1 equiv) and 10 mL DMF were placed in a 50 mL flask and heated at 7O ⁰ C for 16 h, forming a white solid. The solid was mixed with about 25 mL acetonitrile and sonicated until lumps were broken up. The solid was then collected by suction filtration on a medium frit and washed with additional acetonitrile, with a final volume of about 40-50 mL of acetonitrile washings. This helped remove triimidazole monoester E4, which, was not used further in these experiments. The collected solid was transferred to a flask and subject to high vacuum over night, yielding 2.25 g (2.4 mmol) of white solid E5.

Example 13 [0040314-049.1]

N-2-aminoethyl imidazole hydrobromide Al (2.29g, 10 mmol) was suspended in ethanol (30 mL), followed by addition of triethylamine (TEA, approx. 3 mL). The reaction mixture initially became clear and then formed a precipitate. To this mixture was added CF ₃ CO ₂ Et (Fl, approx. 2 mL), and the mixture was stirred at room temperature over night. The precipitate was removed by filtration, and the filtrate was dried by rotoevaporation and high vacuum. The solid residue was suspended in 30 mL THF, Undissolved solid was removed by filtration, and the filtrate was dried by rotoevaporation and high vacuum. The resulting solid was heated in 25-30 mL toluene until it melted, followed by removal of the toluene by rotoevaporation. Addition of toluene was repeated, followed by removal of toluene under high vacuum, yielding 2.0 g (9.6 mmol) F2 in a ratio of about 8:1 relative to residual TEA based on NMR analysis.

Example 14

[0040314-073.1]

F2 (4.17 g, 20 mmol) was dissolved in about 10 mL THF, and an opalescent precipitate was removed by filtration and chased with 10 mL THF, after which bromoacetate phenyl ester F3 (5.16 g, 24 mmol) was added. The mixture was heated to reflux with a heat

gun twice, and was then allowed to stand overnight at room temperature, forming crystals. To this mixture was added 10 mL ether, and the crystals were suspended by sonication, followed by suction filtration and washing with a little THF. The filtered solid was dried under vacuum, yielding 6.85 grams (16.2 mmol) of product F4.

Example 15 [0040314-123.1]

F4 (2.11 g, 5 mmol) was dissolved in acetonitrile (10 mL), producing a hazy solution. To this was added solid N-2-aminoethyl imidazole Al (600 mg, 5.4 mmol) with stirring at room temperature, followed by brief gentle warming, then stirring at room temperature for about 2 h, resulting in formation of adduct FS. Solvent was removed by rotoevaporation at 4O ⁰ C, and 10 mL of acetonitrile was added, followed by bromoacetate phenyl ester F3 (1.11 g, 5.16 mmol) in one portion with stirring at room temperature. The mixture was then warmed gently by heat gun over about 30 min, then rotoevaporated at 4O ⁰ C to form a glass. The glassy material was triturated with 45 mL of THF over night. Solid product was collected by filtration on a medium frit, yielding 2.8 g. This was triturated with 30 mL acetonitrile, warmed gently, then stirred in an ice bath, then warmed to room temperature over 2 h. Solid product was collected by filtration, yielding 1.4 g (2.1 mmol) of F6.

Example 16 [0040314-105.1 - part i]

F4 (1.46 g, 3.5 mmol) was dissolved in about 12 mL of acetonitrile (ACN) with sonication and then was added to a stirred solution of F7 (990 mg, 5.46 mmol) in ACN (2 mL) and stirred at room temperature for about 2 h. Most of the acetonitrile was removed by rotoevaporation (leaving a volume of about 2 mL), and 50 mL ether was added and mixed. The opaque ether layer was decanted, and 20 mL of ether was added, mixed, and then decanted. The acetonitrile layer was dried under high vacuum, producing a sticky foam. The sample was dissolved in aq HBr (10 drops HBr/L of water), applied to a Cl 8 reverse phase silica column (40 by 60 mm, BakerBond Octadecyl 40 Micron Prep LC packing material, PN 7025-01 from J.T. Baker Inc., USA) packed with aq HBr, and eluted with 400 mL of aq HBr, then 220 mL of 200:20 aq HBr/ACN, then 230 mL of 200:30 aq HBr/ACN, then 240 mL of 200:40 aq HBr/ACN. Fractions were analyzed by silica TLC plates and visualized with ninhydrin and/or molybdic acid stain solution (12 g (NH ₄ )SMo ₇ O ₂₄ "4H ₂ O, 0.5 g cerric ammonium nitrate, 50 mL H ₂ SO- _J , and 450 mL water). Product fractions were combined and evaporated under high vacuum to produce F8 as a sticky foam (1.14 g, 2.24 mmol).

Example 17

[0040314-105.1 - part 2]

F§ was dissolved in 15 mL water and 5 mL acetonitrile, 5 mL concentrated NH ₄ OH was then added. After 3 h, solvent was removed by rotoevaporation, the solid residue was dissolved in aq HBr as above and chromatographed on the same column as above (which had been washed with methanol and reequilibrated in aq HBr). Elution was performed using aq HBr, then 100:5 aq HBr/ACN, then 100:10 aq HBr/ACN. Fractions were spotted on silica TLC plates and visualized with niπhydrin. Ninhydrin-reactive fractions were pooled and evaporated. The product was suspended in acetonitrile, and crystals were broken up by sonication. White solid was collected by vacuum filtration on a medium frit and vacuum- dried, yielding 740 mg (1.5 mmol) white solid F9-

Example 18A [0040314-105.1 - part 3]

F9 (11 mg, 22 μmol) was dissolved in 300-500 μL methanol (MeOH) with one drop triethylamine (TEA), and rhodamine dye NHS ester FlQ (11 mg, 16 μmol, see Lee et at, Nucl. Acids Res. 25:2816-2822, 1997) was added as a solid. After 1 h, the mixture was diluted with 0.1% trifluoroacetic acid (TFA) in water and loaded on a reverse phase Cl 8 silica column (10 by 40 mm, from J.T. Baker, supra) that was then eluted with 100:10, 100:20, 100:30, 100:35 0.1% TFA:acetonitrile (110-135 mL each). The colored eluent was collected in 25 mL fractions, each fraction was concentrated and analyzed by HPLC. Fractions 3-7 were pooled and evaporated. Half of the material was submitted for mass spectrometric analysis, and the other half was saved to be used as an HPLC standard. The MS spectrum (MW = 814.73) was consistent with structure FIl.

Example 18B

[0040314-113.1]

As an alternative to the procedure in Example 18 A, F9 (7.4 mg, 15 μmol) was dissolved in a mixture of 140 mg formamide and 5.3 mg TEA. To this was added FlO (8.5 mg, 14.3 μmol) with stirring. HPLC analysis indicated that coupling was complete within about 2 h, forming FIl.

Example 18C

[0040314-109.1]

As a second alternative to the procedure in Example 18A, F9 (7.6 mg, 15.4 μmol) was dissolved in 5% aq NaHCO3 solution (95 mg) and THF (2 drops) was layered on top. FlO

(8.5 mg, 14.3 μmol) was added as a solid in one portion and the mixture was sonicated. HPLC analysis of aliquots (0, 3 h, 5.5 h, and 19 h after sonication) showed a steady increase in the product peak for FIl and a steady decrease in the NHS ester FlO. The reaction was desalted on a small reverse phase column by loading the sample in 0.1% TFA (aq), washing with 10 column volumes of 0.1 % TFA, and eluting with 4:1 acetonitrile:0.1% TFA. After evaporation of solvent, mass spectrometric analysis confirmed that the large peak is product FIl (MW 814.73).

Example 19

[0021112-099]

Gl (4-aminomethyl benzoic acid, 7.5 g, 50 mmol) was suspended in 75 mL DCM, and 10 mL TEA was added, followed by 20 mL trifluoroacetic anhydride (TFAA), and then 10 mL more TEA, so that all reactants dissolved after a while. The mixture was diluted with 500 mL ethyl acetate (EA), washed two times with 1 N HCl (100 mL portions), then two times with 100 mL portions of brine. The organic layer was dried over Na ₂ SO ₄ , filtered, rotoevaporated, and subjected to high vacuum to remove some of the excess trifluoroacetic acid (TFA). The dried material was redissolved in 300 mL 5% NaHCO ₃ solution and washed two times with EA (100 mL portions), acidified with 6 N HCl, and extracted two times with EA (250 mL portions). The combined EA layers were washed two times with brine (100 mL portions), then dried over Na ₂ SO ₄ , filtered, and rotoevaporated. The collected product was then crystallized from 100 mL EA, yielding 6.0 g of crystalline 4- (trifiuoroacetyl)aminomethyl benzoic acid (first crop) and an additional 1.8 g in a second crop (total 7.8 g).

2.45 g of the 4-(trifiuoroacetyl)aminomethyl benzoic acid and 1.4 g of N- hydroxysuccinimide (NHS) were dissolved in 25 mL of THF. To this was added 22 mL of a 0.5 M solution of dicyclohexyl carbodiimide (DCC) in DCM and the mixture was stirred at room temperature. After 45 minutes, precipitated dicyclohexylisourea (DCU) was filtered from the solution, and the solution was diluted with EA to 200 mL final volume. This was washed with two 50 mL portions of 1 N HCl, then with two portions of brine, followed by drying with Na ₂ SO ₄ . The dried solution was filtered, rotoevaporated, and the dried product was crystallized from ethanol, yielding 2.8 g (8.1 mmol) p-(N-trifluoroacetylaminoethyl) benzoate NHS ester G2 as a white solid. Silica TLC eluted with ethyl acetate showed one spot.

Example 20

[0050138-101.1] - part i

4'-aminomethyl fluorescein compound G3 (220 mg, 0.5 mmol) (see Shipchandler et al., Anal. Biochem. 162:89-101 (1987), U.S. Patent No. 4,510,251, and Lee et al., Nucl. Acids Res. 25:2816-2822 (1997)) was dissolved in 5% Na ₂ CO ₃ , forming a dark orange solution, to which was then added about 3 rnL THF. To this mixture was added G2 (200 mg, 0.58 mmol) as a solid. After stirring for 1 h at room temperature, an aliquot (100 μL) was removed and partitioned between ethyl acetate (EA) and 5% HCl (1 mL each). TLC analysis on silica in 5:1 DCM:MeOH indicated that some residual starting material G3 remained at baseline, so another 50 mg (0.14 mmol) of G2 was added to the main reaction mixture and stirred for 30 min more, followed by addition of 5% aqueous HCl to a final volume of 50 mL. This mixture was then extracted with 30 mL EA, then 20 mL EA, and the combined EA layers were washed with two portions of brine and dried over night over Na ₂ SO ₄ . The dried EA solution was filtered and then rotoevaporated. The residue was chromatographed on a silica column (25 by 80 mm) eluted with 10:1 DCM/MeOH containing 1% acetic acid (AA). Product-containing fractions were combined and rotoevaporated and partitioned between EA (100 mL) and 1 N HCl (25 mL), washed with brine (25 mL), and dried over Na ₂ SO ₄ . The EA layer was rotoevaporated and rechromatographed on silica (25 by 80 mm) with 15:1 DCM:M containing 1% AA. All product fractions were combined and rotoevaporated, then respuspended and coevaporated from THF/toluene (5 mL/30 mL) twice, then subject to hi vacuum, yielding 300 mg (0.47 mmol) of product G4.

Example 21

[0050138-101.1] - part 2

Product G4 was dissolved in 6 mL of THF, and 110 mg (1 mmol) of N-hydroxy- succinimide (NHS) was added. The reaction mixture was sonicated, followed by filtration though a plug of glass wool. To the filtered reaction mixture was added 1.4 mL of a 0.5 M DCC/DCM solution (0.7 mmol dicyclohexyl carbodiimide in dichloromethane). After about 35 min, TLC showed very little starting material, so the reaction mixture was filtered through a frit chased with EA, then diluted to about 75 mL with about 25 mL EA, washed with 0.5 N HCl, then twice with brine, and dried over Na ₂ SO.;. The dried solution was filtered, rotoevaporated, reconstituted in EA, sonicated, followed by removal of precipitated DCU by filtration and rotoevaporation of the filtered reaction mixture. The reaction mixture was then chromatographed on a silica column (25 by 80 mm) using 20:1 DCM/MeOH. Fractions were

analyzed by TLC, and product-containing fractions were pooled and rotoevaporated, yielding 350 mg (0.47 mmol) product G5.

Example 22

[0050138-107.1]

Poly-imidazole hexamer C2 (650 mg, 516 μmol, prepared supra) was dissolved in about 4 mL of formamide, then G5 (220 mg, 300 μmol), dissolved in about 5 mL of formamide, was added. The flask containing G5 was washed (chased) two times with one mL portions of formamide into the mixture of C2 and g5, then several drops of TEA were added until the reaction started to become orange. The reactions was allowed to stand at room temp for 2 h and then was analyzed by HPLC (4.6 x 150 mm Cl 8 column, gradient of 5% to 95% B at 1 mL/min over 20 minutes, A = 0.1% aqueous TFA, B = 0.1% TFA in acetonitrile). The largest peak at 9.66 was the product. The reaction mixture was then diluted to about 200 mL with 0.1% aqueous TFA and loaded on a reverse phase silica colun (20 x 60 mm) and eluted with 100:15 0.1% TFA:acetonitrile, then 100:25 , then 100:30, then 100:35 (-200 mL each). Product-containing fractions were analysed by HPLC and the best were pooled, diluted with 2 volumes of 0.1% aq TFA, and trapped on a 10 by 10 mm pad of reverse phase silica, washed with a small volume of 0.1% aq TFA, then eluted with methanol containing about 1 to 2% water. Rotoevaporation and drying under high vacuum yielded 300 mg (147 μmol) of yellow glassy residue G6. This product was dissolved in methanol and split into two 50 mg batches and one 200 mg batch that were also evaporated and placed under high vacuum. HPLC showed a single product peak, and three distinct amide NH hydrogen peaks were observed, consistent with the expected product.

Example 23 Activated Ener ^gy Transfer Dye Conjugate Containing Six Imidazolium Moieties

48 mg (23 μmol) of G6 was dissolved in 3-4 mL MeOH and treated with 1 mL of 10% NaOH for about 2 h. The solution was then diluted with 0.1 % aq trifluoroacetic acid (TFA) to 75 mL, followed by dropwise addition of neat TFA until the pH was about 2. The solution was then passed through a 15 mm by 15 mm plug of reverse phase silica gel (BakerBond Octadecyl 40 Micron Prep LC packing material, PN 7025-01 from J.T. Baker Inc., USA) to trap colored components, then eluted with 100:1 MeOH/H ₂ O containing about 0.3% TFA. The flow of the column was stopped for 10 to 15 min between the collection of each fraction. Removal of solvent from product-containing fractions yielded about 50 mg of deprotected product G7 (having removed the trifluoroacetyl protecting group). This was dissolved in MeOH.

About 80% of G7 solution was stripped and then subjected to high vacuum, yielding 42 mg of G7. This was dissolved in 1 mL of DMF, and 15.5 mg of rhodamine dye NHS ester FlO (supra) was added, followed by about 100 μL of TEA. HPLC of an aliquot after about 25 min indicated that very little starting material remained, and that a large product peak had appeared. After about 2 h, the solution was diluted with aqueous TFA to about 75 mL loaded on a 60 mm by 15 mm reverse phase silica column (J.T. Baker, supra), and eluted with 20:100 (240 mL), 30:100 (260 mL), and 40:100 (280 mL) acetonitrile/aq 0.3% TFA. Product-containing fractions (50 mL each) were identified by HPLC and NMR.

The combined product fractions were diluted 2.5-fold with water and passed through a 10 mm by 15 mm pad of reverse phase Cl 8 silica (J.T. Baker, supra). The trapped compound was washed with 1:10 acetonitrile/aqueous 0.5% TFA and then eluted with 100:1 MeOH/H ₂ O containing about 0.3% TFA. During elution, the flow was stopped for 15 minutes between fractions. The product fractions were concentrated under reduced pressure and then dissolved in 1 mL DMSO (dimethylsulfoxide) and precipitated with 14 mL of ether in a 15 mL Falcon tube. After 2 more precipitations from DMSO/ether, the sample was precipitated twice with DMSO-dg-ether. After removal of the ether by high vacuum, the sample was dissolved in DMSO-dg for NMR analysis, confirming that free acid G8 was obtained.

The G8 product in the NMR sample tube was treated with 10 mg of TSTU (O-(N- Succinimidyl)-l,l,3,3-tetramethyluroniurn tetrafluoroborate) and then with 5 microliters of

triethylamine. After 1 hour, NMR analysis indicated that the desired NHS (N- hydroxysuccinimide) ester G9 had formed. The sample was transferred to a 15 mL Falcon tube with the aid of a small amount of DMSO and precipitated with 13 mL of ethyl acetate. After decantation and vacuum concentration, the sample was dissolved in DMSOd ₆ . NMR analysis showed a singlet at 2.5 ppm integrating as 4 protons, indicating formation of NHS ^ester ω2.- The sample was then precipitated with ether in portions in a 1.5 mL Eppendorf tube and vacuum dried.

Example 24

Nucleotide Synthesis

Compound Hl was dissolved in 50 μL of dry dimethylformamide followed by addition of 15 μL triethylamine. Dye-NHS ester G9 (supra) was added as a solution (15 μL of a 1 mg Dye-NHS ester per 12 μL of DMSO) and stirred in the dark overnight at room temperature. The reaction mixture was purified by cation exchange chromatography (CE- HPLC) (Example 25). Product-containing fractions were concentrated. Final product H2 was dried in vacuo and diluted with 250 mM CAPSO buffer, pH 9.6, to a desired concentration.

Example 25

Nucleotide Syntheses

This example illustrates synthesis of nucleosides (nucleoside triphosphates in this case) containing dye-linker-nucleobase conjugates of the inventions by first attaching a backbone imidazolium linker moiety to a nucleobase of a nucleoside triphosphate and then attaching a dye moiety to the linker (see Scheme 10).

For this example, anion-exchange high-performance chromatography (AE-HPLC) was performed as follows. Column: Aquapore™ AX300, 7 μm particle size and 220 x 4.6 mm (PE Applied Biosystems). Gradient: 30% acetonitrile : 70% triethylammonium bicarbonate (TEAB, 0.1 M) to 30% acetonitrile : 55% TEAB (0.1 M) : 15% TEAB (1.5 M) at 1.5 ml/min over 20 minutes. Detection: UV absorbance at 260 nm or λmax of each dye compound.

Reverse phase high-performance chromatography (RP-HPLC) was performed as follows. Column: RP-C8, 5 μm particle size, 220 x 4.6 mm (Metachem). Solvent A = triethylammonium acetate (TEAA, 0.1 M), Solvent B = TEAA (0.1 M)/acetronitrile 1 :1

Gradient: 10% A : 90% B to 50% A : 50% B at 1.5 ml/min over 10 minutes and then to 100% B over 5 minutes.

Cation-exchange high-performance chromatography (CE-HPLC) was performed as follows. Column: Mono S, 10 x 10 mm (Amersham Biosciences). Gradient: 30% acetonitrile : 70% triethylammonium bicarbonate (TEAB, 0.1 M) to 30% acetonitrile : 70% TEAB (1.5 M) at 3 ml/min over 10 minutes. Detection: UV absorbance at 260 run or λmax of each dye compound.

A. 20 μL of a 30 mM solution of nucleotide triphosphate Hl that contained a reactive amino group on the nucleobase (5-[3-(2-aminoethoxy)propyn-l-yl]-2',3'-dideoxycytidine triphosphate as taught in U.S. Patent No. 6,248,568 to Khan et al.) in 100 mM TEA- bicarbonate (pH 7.0) was evaporated to dryness. It was then dissolved in dry formamide (50 μL) followed by addition of diisopropylethyl amine (20 μL). NHS-ester of a backbone imidazolium linker moiety that contained a TFA-protected amino group (40 μL of a solution of 1 mg linker-NHS ester F4 (supra) per 4 μL of DMSO) was added and stirred over night at room temperature. The reaction mixture was purified by RP-HPLC. Fractions corresponding to NTP product H3 were concentrated to dryness.

B. The trifluoroacetyl protecting group was removed by treatment with 200-300 μL of ammonium hydroxide solution (28-30%) at 55 ⁰ C for 30 minutes. Concentration under vacuum gave deprotected compound H4 which was purified by RP-HPLC.

C. All of H4 was dissolved in 50 μL of dry formamide followed by addition of 5 μL diisopropylethyl amine. Dye-NHS ester FJj) (supra) was added as a solid (2 mg) and stirred in the dark overnight at room temperature. The reaction mixture was purified by anion exchange chromatography (AE-HPLC). Product-containing fractions were concentrated and repurified by RP-HPLC. Final product H5 was dried in vacuo and diluted witib 250 mM CAPSO buffer, pH 9.6, to a desired concentration.

D. As an alternative to paragraph C, the following procedure can be used to form nucleotide triphosphates containing energy transfer dyes. Energy transfer dye (1 mg) was dissolved in 30 μL DMSO followed by addition of triethylamine (5 μL) and TSTU (1 mg). The reaction mixture was stirred at room temperature for 30 min. 20 μL of this reaction mixture was added to ddNTP Hl (0.3 μmol in 50 μL DMSO) that contained a reactive amino group on the nucleobase, and the reaction mixture was stirred for 1 h at room temperature. The reaction mixture was purified by cation exchange chromatography (CE-HPLC).

Product-containing fractions were dried in vacuo and diluted with 100 mM TEAB, pH 8.0, to a desired concentration.

Although the present disclosure is illustrated with respect to certain embodiments and examples, it will be appreciated that various modifications and variations can be made without departing from the scope and spirit of the inventions described herein.