Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-08-01
2004-02-03
Wilson, James O. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S022100, C536S025300, C536S025320
Reexamination Certificate
active
06686461
ABSTRACT:
FIELD OF THE INVENTION
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.
BACKGROUND OF THE INVENTION
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.
SUMMARY OF THE INVENTION
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 (—O—NH
2
), 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
(3
rd 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 hydrazi
Hogrefe Richard I.
Schwartz David A.
Heller Ehrman White & McAuliffe LLP
Lewis Patrick
Solulink Bioscience, Inc.
Wilson James O.
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