Monomers and methods for preparation, detection and immobilization of macromolecules, including biopolymers, and for preparation and detection of macromolecular conjugates are provided. The monomers for use in the methods provided herein include hydrazino, oxyamino and carbonyl substituted nucleoside triphosphates. Biopolymers, including oligonucleotides, possessing hydrazino, oxyamino or carbonyl modifications are also provided.
Polymerase chain reaction (PCR) expression of oligonucleotides is a powerful tool in molecular biology. The PCR product is generally labelled to aid in detection. Various methods of direct and indirect labelling of the PCR product have been developed. Direct labelling involves the incorporation of a labelled monomer during enzymatic synthesis, while indirect labelling refers to post-synthetic introduction of the label.
For example, methods for direct labelling of PCR products are known (see, U.S. Pat. No. 5,242,756 and International Patent Application Publication No. WO 99/65993) In such methods, modified nucleoside triphosphates are incorporated into oligonucleotides during during amplification; and conjugation of a xanthene or cyanine label to a nucleoside triphosphate is effected via an amide bond formed with the modified nucleoside triphosphates. Other methods involve conjugation of a biotin or metal chelating label to an oligonucleotide via an amide or hydrazide bond is used to forme a desired oligonucleotide-label conjugates (see, U.S. Pat. Nos. 4,707,440 and 4,889,798). Another method for indirect labelling of oligonucleotides involves incorporation of a boronic acid containing nucleoside triphosphate into an oligonucleotide during enzymatic synthesis. Complexation of this boronic acid modified oligonucleotide with a hydroxamic acid derivatized label provides the desired labelled oligonucleotide (see, U.S. Pat. No. 5,876,938).
These methods of oligonucleotide labelling, however, are limited by the lack of stability of the label to the amplification reaction conditions, the inability to selectively and specifically incorporate multiple labels, and the instability of succinimidyl esters. Preparation of activated functionalities, such as succinimidyl esters or maleimides, of labels can be costly, and not even possible in some instances, particularly under PCR conditions.
Thus, due to the limitations of currently available methods as described above, there is a need for efficient methods for labelling of oligonucleotides. Therefore, it is an object herein to provide monomers and methods for labelling of oligonucleotides without the need for post-synthetic modification of the oligonucleotide. It is also an object herein to provide monomers and methods for enzymatic synthesis of modified biopolymers, including oligonucleotides, that can be specifically labelled. A further object herein is to provide the resulting modified oligonucleotides.
Oligonucleotide monomers containing hydrazino, oxyamino, or carbonyl groups that can be incorporated into an oligonucleotide chain during enzymatic oligonucleotide synthesis are provided. Methods for immobilization and conjugation of biopolymer first components, particularly oligonucleotides, containing hydrazino, oxyamino, or carbonyl modifications are provided. The monomers are triphosphate nucleoside derivatives that can be incorporated into an oligonucleotide first component during enzymatic synthesis, including, but not limited to, synthesis by polymerase chain reactions (PCR), reverse trascriptases, including, but not limited to, AMV reverse transcriptase, MMLV reverse transcriptase and superscript reverse transcriptase, and polymerases, including, but not limited to, Taq polymerase, DNA plymerase, Klenow fragment and T4 DNA polymerase. The resulting first components can then be used for any purpose for which oligonucleotides are used. They are particularly suitable for conjugation to a second component or immobilizion on a surface. The monomers provided herein advantageously are readily incorporated into oligonucleotide chains, hence can be used in any application that involves or uses a nucleoside triphosphate, such as DNA and RNA sequencing, detecting, labeling and amplification methodologies. Monophosphate and diphoshate forms of the monomers as well as nucleic acid chains containing the incorporated monomers are provided.
The monomers provided herein are also useful as mass modifiers in DNA sequencing by mass spectrometry (see, e.g., U.S. Pat. Nos. 6,074,823 and 5,547,835). The monomers can be incorporated into oligonucleotides for the accurate determination of base composition (Muddiman et al. (1997) Anal. Chem. 69:1543), and for the scoring of single nucleotide polymorphisms (SNPs) (Chen et al. (1999) Anal. Chem. 71:3118). The monomers can also be used to study the mechanisms by which ribozymes effect catalytic cleavage (Earnshaw et al. (2000) Biochemistry 39:6410). The monomers can be incorporated into antisense oligonucleotides to increase their resistance to enzymatic degradation (Verheijen et al. (2000) Bioorg. Med. Chem. Lett. 10:801), their overall potency (Flanagan et al. (1999) Proc. Natl. Acad. Sci. USA 96:3513) and the stability of their hybrids with the complementary RNA sequences (Compagno et al. (1999) J. Biol. Chem. 274:8191).
The monomers possess a triphosphate-ribose-nucleobase motif for recoginition by the enzymatic catalyst.
Riboses for use in the monomers and methods herein are well known to those of skill in the art, and include, but are not limited to, fully hydroxylated sugars such are ribose, deoxyriboses such as 2-deoxyriboses, and dideoxy riboses such as 2,3-dideoxyribose.
Nucleobases for use in the monomers and methods herein are also well known to those of skill in the art, and include, but are not limited to, cytosines, uracils, adenines, guanines and thymines, and analogs thereof, including deaza analogs.
The monomers also possess, in addition to the triphosphate group, a protected or unprotected hydrazino, protected or unprotected oxyamino (xe2x80x94Oxe2x80x94NH2), or carbonyl moiety for formation of a hydrazone or oxime linkage with an appropriately modified surface or second component. The hydrazino moiety can be an aliphatic, aromatic or heteroaromatic hydrazine, semicarbazide, carbazide, hydrazide, thiosemicarbazide, thiocarbazide, carbonic acid dihydrazine or hydrazine carboxylate (see, FIG. 1). The protecting groups are salts of the hydrazino or oxyamino group, including but not limited to, mineral acids salts, such as but not limited to hydrochlorides and sulfates, and salts of organic acids, such as but not limited to acetates, lactates, malates, tartrates, citrates, ascorbates, succinates, butyrates, valerates and fumarates, or any amino or hydrazino protecting group known to those of skill in the art (see, e.g., Greene et al. (1999) Protective Groups in Organic Synthesis (3rd Ed.) (J. Wiley Sons, Inc.)). The carbonyl moiety can be any carbonyl containing group capable of forming a hydrazone or oxime linkage with one or more of the above hydrazino or oxyamino moieties. Preferred carbonyl moieties include aldehydes and ketones.
Second components include, but are not limited to, macromolcules, biopolymers as defined herein, polymers including, but not limited to, polyamines, polyamides, polyethers and polyethylene glycols, and other compounds of interest herein for use in assays, kits, diagnostic arrays, and the like, including, but not limited to, intercalators, vitamins, reporter molecules, cholesterols, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, dyes, antibodies, haptens, antigens, enzymes, and detection reagents including, but not limited to, fluorophores, metals including, but not limited to, gold, metal chelates, chromophores, fluorophore precursors and chromophore precursors, that possess or are modified to possess a hydrazino, oxyamino or carbonyl group that is complementary to the carbonyl, oxyamino or hydrazino group of the oligonucleotide of formula (II) for formation of hydrazone or oxime linkage. Dendrimeric compounds that possess a plurality of detectable groups, including, but not limited to, reporter molecules, fluorophores, chromophores, fluorophore precursors and chromophore precursors, are also contemplated herein as second components. Fluorophore precursors and chromophore precursors are compounds that react with the hydrazino, oxyamino or carbonyl group of the modified oligonucleotide to form a fluorogenic or chromogenic group for analysis. Such groups are preferred due to the absence of background noise in the resulting assay. Preferred conjugates include those containing a hydrazone linkage.
In one embodiment, the monomers for use in the methods provided herein are capture nucleoside triphosphates (cNTPs) that have formula (I):
P1xe2x80x94S1xe2x80x94B1xe2x80x94Mxe2x80x94X
or a dervative thereof, as defined herein, where P1 is a triphosphate group, as defined herein; S1 is a ribose, a deoxyribose or a dideoxyribose; B1 is a nucleobase; X is a protected or unprotected hydrazino group, a protected or unprotected oxyamino group, or a carbonyl derivative, where the protecting group is a salt or any amino or hydrazino protecting group known to those of skill in the art; and M is a divalent group having any combination of the following groups, which can be combined in any order: arylene, heteroarylene, cycloalkylene, C(R1)2, xe2x80x94C(R1)xe2x95x90C(R1)xe2x80x94,  greater than Cxe2x95x90C(R2)(R3),  greater than C(R2)(R3), xe2x80x94Cxe2x89xa1Cxe2x80x94, O, S(A)a, P(D)b(R1), P(D)b(ER1), N(R1),  greater than N+(R2)(R3) and C(E); where a is 0, 1 or 2; b is 0, 1, 2 or 3; A is O or NR1; D is S or O; and E is S, O or NR1; each R1 is a monovalent group independently selected from hydrogen and M1xe2x80x94R4; each M1 is a divalent group independently having any combination of the following groups, which groups can be combined in any order: a direct link, arylene, heteroarylene, cycloalkylene, C(R5)2, xe2x80x94C(R5)xe2x95x90C(R5)xe2x80x94,  greater than Cxe2x95x90C(R2)(R3),  greater than C(R2)(R3), xe2x80x94Cxe2x89xa1Cxe2x80x94, O, S(A)a, P(D)b(R5), P(D)b(ER5), N(R5), N(COR5),  greater than N+(R2)(R3) and C(E); where a is 0, 1 or 2; b is 0, 1, 2 or 3; A is O or NR5; D is S or O; and E is S, O or NR5; R4 and R5 are each independently selected from among hydrogen, halo, pseudohalo, cyano, azido, nitro, SiR6 R7R8, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR9R10; R9 and R10 are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl and heterocyclyl; R2 and R3 are selected from (i) or (ii) as follows: (i) R2 and R3 are independently selected from among hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heteroaryl; or (ii) R2 and R3 together form alkylene, alkenylene or cycloalkylene; R6, R7 and R8 are each independently a monovalent group selected from hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR9R10; and
R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10 are optionally substituted with one or more substituents each independently selected from Z, wherein Z is selected from alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, hydroxy, S(O)hR20, NR20R21, COOR20, COR20, CONR20R21, OC(O)NR20R21, N(R20)C(O)R21, alkoxy, aryloxy, heteroaryl, heterocyclyl, heteroaryloxy, heterocyclyloxy, aralkyl, aralkenyl, aralkynyl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, aralkoxy, heteroaralkoxy, alkoxycarbonyl, carbamoyl, thiocarbamoyl, alkoxycarbonyl, carboxyaryl, halo, pseudohalo, haloalkyl and carboxamido; h is 0, 1 or 2; and R20 and R21 are each independently selected from among hydrogen, halo, pseudohalo, cyano, azido, nitro, trialkylsilyl, dialkylaryisilyl, alkyldiarylsilyl, triarylsilyl, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy, amino, amido, alkylamino, dialkylamino, alkylarylamino, diarylamino and arylamino.
Macromolecutions, particularly biopolymers, including oligonucleotides, that are modified by incorporation of the above monomers are provided. Thus, in certain embodiments, provided herein are oligonucleotide analogs of formula (II): 
or a derivative thereof, where O1 and O2 are each independently oligonucleotides or analogs thereof, such as protein nucleic acids (PNAs); P2 is a phosphodiester group, resulting from the coupling of a compound of formula (I) with O1 or analog thereof; and S1, B1, M and X are selected as above.
Such oligonucleotide analogs are useful as antisense drugs, cis-lements acting as regulators of gene expression, and substrates for RNA binding proteins such as HIV-1 Rev and Tat (see, eq., Agrawal (1999) Biochim. Biophys. Acta 1489:53; Crooke (1999) Biochim. Biophys. Acta 1489:31; Gryaznov (1999) Biochim. Biophys. Acta 1489:131; and Morishita (2000) Nipp. Yakuri. Zass. 115:123).
Oligonucleotides covalently immobilized on a solid surface, as described herein, are also provided. The oligonucleotides are of formula (II), and are immobilized through a covalent hydrazone or oxime linkage. The solid surface is modified to possess a hydrazino, oxyamino, or carbonyl group that is complementary to the hydrazino, oxyamino or carbonyl moiety of the oligonucleotide of formula (II) for formation of a hydrazone or oxime linkage. In preferred embodiments, the oligonucleotides are immobilized through a covalent hydrazone linkage.
The immobilized oligonucleotides, as well as non-immobilized forms, are useful for a variety of purposes known to those of skill in the art, including, but not limited to, preparation of microarrays, diagnostic probe assays, DNA or RNA amplification, for example by solid phase polymerase chain reactions (PCR), molecular computing (see, e q, Adleman (1994) Science 266:1021-1024; Kari (1997) Mathematical Intelligencer 19:9-22; Frutos et al. (1997) Nucleic Acids Res. 25:4748; Smith et al. (1998) J. Comp. Biol. 5:255; Liu et al. (1998) J. Comp. Biol. 5:267; Frutos et al. (1998) J. Am. Chem. Soc. 120:10277; Wang et al. (1999) Biosystems 52:189-191; Liu et al. (1999) Biosystems 52:25-33; Liu et al. (2000) Nature 403:175-179; European Patent Application Publication No. EP 0 772 135; Reed et al. (June 2000) Scientific American:86-93), molecular addressing (Niemeyer et al. (1994) Nucl. Acids Res. 22(25):5530-5539), DNA and RNA sequencing methods, including mass spectrometry methods (see, e.g., U.S. Pat. Nos. 6,074,823 and 5,547,835), nucleic acid diagnositics, including SNP and other polymorphism analyses and detection methods, and in studying the molecular electronics of DNA (see, e.g., U.S. Pat. Nos. 6,071,699, 6,066,448, 5,952,172 and 5,824,473).
The oligonucleotides can also be used in PCR-based sequencing methods, particularly solid phase sequencing methods using the immobilized oligonucleotides (Mustajoki et al. (1997) Genome Res. 7:1054). They can also be used to measure the interaction forces between single strands of DNA (Lee et al. (1994) Science 266:771), in solid phase-mediated transfection of oligonucleotides (Bielinska et al. (2000) J. Biomaterials 21:877), and in solid phase cloning to create libraries suitable for direct solid phase sequencing (Hultman et al. (1994) J. Biotechnol. 35:229). The immobilized oligonucleotides can also be used in DNA chip technology to create arrays of oligonucleotides which are used to compare the qualitative and quantitative characteristics of gene expression profiles, mutations, insertions and deletions in normal and diseased states (De Benedetti et al. (2000) Int. J. Biol. Markers 15:1). They can also be used to identify and characterize the DNA binding site of DNA binding proteins (Roth et al. (1995) EMBO J. 14:2106; Carlsson et al. (1995) Anal. Biochem. 232:172). Immobilized oligonucleotides bound to Sephacryl S-500 particles via a CNBr-activation procedure can be used to assemble extended DNA duplexes by phosphorylation, ligation and restriction enzyme digestion of assemblies of annealed oligonucleotides in solid phase (Hostomsky et al. (1987) Nucleic Acids Symp. Ser. 18:241). The immobilized oligonucleotides can also be used in PCR, RT-PCR (Kozwich et al. (2000) Appl. Environ. Microbiol. 66:2711; Blomqvist et al. (1999) J. Clin. Microbiol. 37:2813), transcription (Marble et al. (1995) Biotechnol. Prog. 11:393; Fujita et al. (1993) Biotechniques 14:608), ligation reactions (Filippov et al. (1990) Bioorg. Khim. 16:1045), and in studying DNA repair mechanisms (Salles et al. (1999) Biochimie 81:53).
In embodiments where X of the modified oligonucleotide is a hydrazino or oxyamino group, the solid surface can be modified to possess an epoxide, xcex1-bromocarbonyl, maleimide, maleic anhydride, isothiocyanate or isocyanate group. Such solid surfaces can be prepared by methods provided herein or other methods well known to those of skill in the art. For example, reaction of pentafluorophenyl 4-isothiocyanato-benzoate with an amino solid surface results in formation of an isothiocyanato modified solid surface. Some of these surfaces are commercially available from, e.g., Pierce (Rockford, Ill.), SINTEF Applied Chemistry (Trondheim, Norway), Rapp Polymere Gmbh (Tubingen, Germany), and Dyno Particles AS (Trondheim, Norway). Reaction of the hydrazino or oxyamino group of the modified oligonucleotide with the epoxide, xcex1-bromocarbonyl, maleimide, maleic anhydride, isothiocyanate or isocyanate group of the solid surface results in covalent attachment of the oligonucleotide to the solid surface.
In certain embodiments, particularly where X is an oxyamino group, the immobilized oligonucleotides are selected such that the solid surface is not modified with an aldehyde or epoxide group.
Oligonucleotide conjugates are provided. The conjugates are prepared from the modified oligonucleotide first components of formula (II). The modified oligonucleotide of formula (II) is reacted with a complementary derivative of a second component to form a hydrazone or oxime covalent linkage. The second components include, but are not limited to, biopolymers as defined herein, polymers including, but not limited to, polyamines, polyamides, polyethers and polyethylene glycols, and other compounds of interest herein for use in assays, kits, diagnostic arrays, and the like, including, but not limited to, intercalators, vitamins, reporter molecules, cholesterols, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, dyes, antibodies, haptens, antigens, enzymes, and detection reagents including, but not limited to, fluorophores, metals including, but not limited to, gold, metal chelates, chromophores, fluorophore precursors and chromophore precursors, that possess or are modified to possess a hydrazino, oxyamino or carbonyl group that is complementary to the carbonyl, oxyamino or hydrazino group of the oligonucleotide of formula (II) for formation of hydrazone or oxime linkage. Dendrimeric compounds that possess a plurality of detectable groups, including, but not limited to, reporter molecules, fluorophores, chromophores, fluorophore precursors and chromophore precursors, are also contemplated herein as second components. Fluorophore precursors and chromophore precursors are compounds that react with the hydrazino, oxyamino or carbonyl group of the modified oligonucleotide to form a fluorogenic or chromogenic group for analysis. Such groups are preferred due to the absence of background noise in the resulting assay. Preferred conjugates include those containing a hydrazone linkage.
In embodiments where X is a hydrazino or oxyamino group, the second component can be modified to possess an epoxide, xcex1-bromo-carbonyl, maleimide, maleic anhydride, isothiocyanate or isocyanate group. Such second components can be prepared by methods provided herein or by other methods well known to those of skill in the art. For example, reaction of pentafluorophenyl 4-isothiocyanatobenzoate with an amino or hydroxy group of a second component, such as a protein or oligosaccharide, results in formation of an isothiocyanato modified second component. Reaction of the hydrazino or oxyamino group of the modified oligonucleotide with the epoxide, xcex1-bromocarbonyl, maleimide, maleic anhydride, isothiocyanate or isocyanate group of the second component results in covalent attachment of the oligonucleotide to the second component to form the conjugates provided herein.
In certain embodiments herein, the oligonucleotide conjugates are selected with the proviso that the covalent linkage is not an acyl hydrazone (xe2x80x94C(O)NHNHxe2x95x90). In preferred embodiments, the oligonucleotide conjugates are prepared from modified oligonucleotides of formula (II) where P2 is not a phosphoramidate. In other embodiments, the oligonucleotide conjugates are selected with the proviso that the second component is not a protein, particularly a glycoprotein, more particularly an immunoglobulin.
The modified oligonucleotides provided herein can also be conjugated to ligands such as growth factors, membrane-active bacterial proteins and asialoglycoproteins as delivery strategies for antisense therapy and gene therapy (see, e.g, Perales et al. (1994) Eur. J. Biochem. 226:255; Cristiano et al. (1996) Cancer Gene Ther. 3:4; Hoganson et al. (1998) Hum. Gene Ther. 9:2565; Gottschalk et al. (1995) Gene Ther. 2:498; Lu et al. (1994) J. Nucl. Med. 35:269). Oligonucleotide-antigen conjugates can be used as immunomodulators in regulating airway eosinophilia in bronchial asthma (Shirota et al. (2000) J. Immunol. 164:5575). Oligonucleotide probes conjugated to fluorescent molecules are used in fluorescence in situ hybridization (FISH) for chromosome classification and the detection of chromosome aberrations (Pinkel et al. (1986) Proc. Natl. Acad. Sci. USA 83:2934). Oligonucleotide conjugates are also used to study the mechanics of DNA hybridization, and to investigate protein-DNA contacts of DNA binding proteins (Lannutti et al. (1996) Biochemistry 35:9821; Brown et al.(1997) Curr. Opin. Biotechnol. 8:45).
Importantly, the immobilized oligonucleotides or oligonucleotide conjugates provided herein can be formed under aqueous conditions without the need for additional reagents, such as a reducing agent.
Methods for the attachment of hydrazino, oxyamino, or carbonyl modified oligonucleotides of formula (II) to appropriately modified surfaces, as described herein, are provided. The attachment is via a covalent hydrazone or oxime bond formed from the hydrazino, oxyamino, or carbonyl group of the modified oligonucleotide and a complementary carbonyl, oxyamino or hydrazino modified surface provided herein (see, FIG. 2).
Methods for the conjugation of oligonucleotide first components to second components, including, but not limited to, biopolymers as defined herein, polymers including, but not limited to, polyamines, polyamides, polyethers and polyethylene glycols, and other compounds of interest herein for use in assays, kits, diagnostic arrays, and the like, including, but not limited to, intercalators, vitamins, reporter molecules, cholesterols, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, dyes, antibodies, haptens, antigens, enzymes, and detection reagents including, but not limited to, fluorophores, metals including, but not limited to, gold, metal chelates, chromophores, fluorophore precursors and chromophore precursors; that possess or are modified to possess a hydrazino, oxyamino or carbonyl group that is complementary to the carbonyl, oxyamino or hydrazino group of the oligonucleotide of formula (II) for formation of hydrazone or oxime linkage, are provided. Dendrimeric compounds that possess a plurality of detectable groups, including, but not limited to, reporter molecules, fluorophores, chromophores, fluorophore precursors and chromophore precursors, are also contemplated herein as second components. Fluorophore precursors and chromophore precursors are compounds that react with the hydrazino, oxyamino or carbonyl group of the modified oligonucleotide to form a fluorogenic or chromogenic group for analysis.
In particular, methods for conjugation of a hydrazino, oxyamino, or carbonyl modified oligonucleotide of formula (II) with an appropriately modified second component are provided. The conjugation is achieved through a covalent hydrazone or oxime bond formed from the hydrazino, oxyamino or carbonyl group of the modified oligonucleotide and a second component possessing a complementary carbonyl, oxyamino or hydrazino group.
In all embodiments herein, it is preferred that one, more preferably both, of the reactive partners (eq., the hydrazino, oxyamino or carbonyl groups) are aromatic or heteroaromatic. Thus, in preferred embodiments, the compounds of formula (I) will be aryl or heteroaryl hydrazino or oxyamino derivatives, or aryl or heteroaryl carbonyl derivatives. In more preferred embodiments, the coupling partner (e.g., the modified solid surface or the second component) will also possess an aryl or heteroaryl hydrazino or oxyamino group, or an aryl or heteroaryl carbonyl group. Hydrazone and oxime linkages formed from these preferred groups are more stable than the corresponding aliphatic hydrazone and oxime linkages, and thus are more preferred in certain applications.
A composition, comprising one or more nucleoside triphosphates and a monomer provided herein. Methods using the composition to synthesize nucleic acid molecules that include the monomers provided herein incoporated into the chains. Nucleic acid molecules (i.e. oligonucleotides) in which the monomers provided herein are at the end are also provided; these are produced by providing the monomers as chain terminators, such as dideoxynucleotides.
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