Nucleoside triphosphates and their incorporation into...

Details Nucleoside triphosphates and their incorporation into... Nucleoside triphosphates and their incorporation into...

1999-04-28

2002-11-19

Richter, Johann (Department: 1623)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S023200, C536S025100, C536S025340, C514S04400A, C435S009000

Reexamination Certificate

active

06482932

ABSTRACT:

BACKGROUND OF THE INVENTION
This invention relates to novel nucleotide triphosphates (NTPs); methods for synthesizing nucleotide triphosphates; and methods for incorporation of novel nucleotide triphosphates into oligonucleotides. The invention further relates to incorporation of these nucleotide triphosphates into nucleic acid molecules using polymerases under several novel reaction conditions.
The following is a brief description of nucleotide triphosphates. This summary is not meant to be complete, but is provided only to assist understanding of the invention that follows. This summary is not an admission that all of the work described below is prior art to the claimed invention.
The synthesis of nucleotide triphosphates and their incorporation into nucleic acids using polymerase enzymes has greatly assisted in the advancement of nucleic acid research. The polymerase enzyme utilizes nucleotide triphosphates as precursor molecules to assemble oligonucleotides. Each nucleotide is attached by a phosphodiester bond formed through nucleophilic attack by the 3′ hydroxyl group of the oligonucleotide's last nucleotide onto the 5′ triphosphate of the next nucleotide. Nucleotides are incorporated one at a time into the oligonucleotide in a 5′ to 3′ direction. This process allows RNA to be produced and amplified from virtually any DNA or RNA templates.
Most natural polymerase enzymes incorporate standard nucleotide triphosphates into nucleic acid. For example, a DNA polymerase incorporates dATP, dTTP, dCTP, and dGTP into DNA and an RNA polymerase generally incorporates ATP, CTP, UTP, and GTP into RNA. There are however, certain polymerases that are capable of incorporating non-standard nucleotide triphosphates into nucleic acids (Joyce, 1997
, PNAS
94, 1619-1622, Huang et al.,
Biochemistry
36, 8231-8242).
Before nucleosides can be incorporated into RNA transcripts using polymerase enzymes they must first be converted into nucleotide triphosphates which can be recognized by these enzymes. Phosphorylation of unblocked nucleosides by treatment with POCl
3
and trialkyl phosphates was shown to yield nucleoside 5′-phosphorodichloridates (Yoshikawa et al., 1969
, Bull. Chem. Soc
.(Japan) 42, 3505). Adenosine or 2′-deoxyadenosine 5′-triphosphate was synthesized by adding an additional step consisting of treatment with excess tri-n-butylammonium pyrophosphate in DMF followed by hydrolysis (Ludwig, 1981
, Acta Biochim. et Biophys. Acad. Sci. Hung
. 16, 131-133).
Non-standard nucleotide triphosphates are not readily incorporated into RNA transcripts by traditional RNA polymerases. Mutations have been introduced into RNA polymerase to facilitate incorporation of deoxyribonucleotides into RNA (Sousa & Padilla, 1995
, EMBO J
. 14,4609-4621, Bonner et al., 1992
, EMBO J
. 11, 3767-3775, Bonner et al., 1994
, J. Biol. Chem
. 42, 25120-25128, Aurup et al., 1992
, Biochemistry
31, 9636-9641).
McGee et al., International PCT Publication No. WO 95/35102, describes the incorporation of 2′-NH
2
-NTP's, 2′-F-NTP's, and 2′-deoxy-2′-benzyloxyamino UTP into RNA using bacteriophage T7 polymerase.
Wieczorek et al., 1994
, Bioorganic
&
Medicinal Chemistry Letters
4, 987-994, describes the incorporation of 7-deaza-adenosine triphosphate into an RNA transcript using bacteriophage T7 RNA polymerase.
Lin et al., 1994
, Nucleic Acids Research
22, 5229-5234, reports the incorporation of 2′-NH
2
-CTP and 2′-NH
2
-UTP into RNA using bacteriophage T7 RNA polymerase and polyethylene glycol containing buffer. The article describes the use of the polymerase synthesized RNA for in vitro selection of aptamers to human neutrophil elastase (HNE).
SUMMARY OF THE INVENTION
This invention relates to novel nucleotide triphosphate (NTP) molecules, and their incorporation into nucleic acid molecules, including nucleic acid catalysts. The NTPs of the instant invention are distinct from other NTPs known in the art. The invention further relates to incorporation of these nucleotide triphosphates into oligonucleotides using an RNA polymerase; the invention further relates to novel transcription conditions for the incorporation of modified (non-standard) and unmodified NTP's into nucleic acid molecules. Further, the invention relates to methods for synthesis of novel NTP's
In a first aspect, the invention features NTP's having the formula triphosphate-OR, for example the following formula I:
where R is any nucleoside; specifically the nucleosides 2′-O-methyl-2,6-diaminopurine riboside; 2′-deoxy-2′amino-2,6-diaminopurine riboside; 2′-(N-alanyl)amino-2′-deoxy-uridine; 2′-(N-phenylalanyl)amino-2′-deoxy-uridine; 2′-deoxy-2′-(N-&bgr;-alanyl)amino; 2′-deoxy-2′-(lysiyl)amino uridine; 2′-C-allyl uridine; 2′-O-amino-uridine; 2′-O-methylthiomethyl adenosine; 2′-O-methylthiomethyl cytidine; 2′-O-methylthiomethyl guanosine; 2′-O-methylthiomethyl-uridine; 2′-Deoxy-2′-(N-histidyl)amino uridine; 2′-deoxy-2′-amino-5-methyl cytidine; 2′-(N-&bgr;-carboxamidine-&bgr;-alanyl)amino-2′-deoxy-uridine; 2′-deoxy-2′-(N-&bgr;-alanyl)-guanosine; 2′-O-amino-adenosine; 2′-(N-lysyl)amino-2′-deoxy-cytidine; 2′-Deoxy-2′-(L-histidine)amino Cytidine; and 5-Imidazoleacetic acid 2′-deoxy-5′-triphosphate uridine. In a second aspect, the invention features a process for the synthesis of pyrimidine nucleotide triphosphate (such as UTP, 2′-O-MTM-UTP, dUTP and the like) including the steps of monophosphorylation where the pyrimidine nucleoside is contacted with a mixture having a phosphorylating agent (such as phosphorus oxychloride, phospho-tris-triazolides, phospho-tris-triimidazolides and the like), trialkyl phosphate (such as triethylphosphate or trimethylphosphate or the like) and dimethylaminopyridine (DMAP) under conditions suitable for the formation of pyrimidine monophosphate; and pyrophosphorylation where the pyrimidine monophosphate is contacted with a pyrophosphorylating reagent (such as tributylammonium pyrophosphate) under conditions suitable for the formation of pyrimidine triphosphates.
The term “nucleotide” as used herein is as recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1′ position of a sugar moiety. Nucleotides generally include a base, a sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No. WO 92/07065; Usman et al., International PCT Publication No. WO 93/15187; all hereby incorporated by reference herein). There are several examples of modified nucleic acid bases known in the art as recently summarized by Limbach et al., 1994
, Nucleic Acids Res
. 22, 2183. Some of the non-limiting examples of base modifications that can be introduced into nucleic acids without significantly effecting their catalytic activity include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine) and others (Burgin et al., 1996
, Biochemistry
, 35, 14090). By “modified bases” in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1′ position or their equivalents; such bases may be used within the catalytic core of an enzymatic nucleic acid molecule and/or in the substrate-binding regions of such a molecule. Such modified nucleotides include dideoxynucleotides

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