Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-11-16
2002-06-04
Riley, Jezia (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S091200, C536S022100, C536S023100, C536S025300
Reexamination Certificate
active
06399335
ABSTRACT:
FIELD OF THE INVENTION
The field of the invention is the use of &ggr;-phosphoester nucleoside triphosphates in polymerase reactions.
BACKGROUND OF THE INVENTION
The human immunodeficiency virus infects over 40 million people worldwide and the Hepatitis C virus has infected approximately 2% of the world's population. The economic and medical impacts of such emerging epidemics demonstrate clearly the need to rapidly and effectively assess the efficacy of viral inhibitors. Assaying inhibitors of viral RNA or DNA synthesis is a time consuming and costly process that often requires radioisotopes. This screening process is expensive, time consuming, and requires special handling due to the use of radioisotopes. Previous attempts to make colorimetric or fluorescent nucleotide analogs useful for the detection of polymerase activity primarily employ a detectable chromophore or fluorophore attached to the base or ribose portion of a nucleotide. The signal is then incorporated into the newly formed product nucleic acid, hence necessitating an often lengthy and labor-intensive step to separate the products from the reactants.
Several companies sell products that incorporate a detectable reagent into the product of polymerase synthesis, including Boehringer (Genius kit), Life Technologies INC., GIBC/BRL, Sigma (biotinylated nucleotides, fluorescent nucleotides), Molecular Probes Inc. (a large range of fluorescent and caged nucleotides), Li-Cor (dyes attached to DNAs for DNA sequencing), etc. Reports of &ggr;-phosphoesters of nucleoside triphosphates have described them as non-hydrolyzable and used them in solid phase affinity purification protocols, e.g. Clare M. M. Haystead, et al., Gamma-phosphate-linked ATP-Sepharosefor the affinity purification of protein kinases, Eur. J. Biochem. 214, 459-467 (1993), esp. p.460, col. 2, line 23. We synthesized large numbers of &ggr;-phosphoester nucleoside triphosphates and found that while they are indeed non-hydrolyzable by many enzymes, they are often suitable substrates for RNA and DNA polymerases.
SUMMARY OF THE INVENTION
The invention provides methods and compositions for polymerizing a particular nucleotide with a polymerase. In general, the method involves (a) forming a mixture of a polymerase and a nucleoside triphosphate (NTP) comprising &agr;, &bgr; and &ggr; phosphates and a &ggr;-phosphate phosphoester-linked functional group; and (b) incubating the mixture under conditions wherein the polymerase catalyzes cleavage of the NTP between the &agr; and &bgr; phosphates, liberating a pyrophosphate comprising the functional group and polymerizing the resultant nucleoside monophosphate. i.e. incorporates the nucleoside monophosphate in a nascent polynucleotide.
A variety of functional groups compatible with the polymerization reaction are provided. In one embodiment, the functional group is a detectable label and the method further comprises the step of detecting the label, wherein a wide variety of chromogenic and luminogenic labels are provided.
In another embodiment, the functional group is a cell delivery enhancing moiety, —OR, wherein R is independently selected from: substituted or unsubstituted (C1-C18) alkyl, alkenyl, alkynyl and aryl, each inclusive of carbocyclic and heterocyclic. These substituents provide enhanced therapeutic availability through enhanced gut or blood stability, cellular and/or membrane permeability, host phosphatase stability, etc. This aspect provides a wide variety of generally membrane permeable, relatively hydrophobic R substituents.
In another embodiment, the functional group is a polymerase specificity enhancing moiety, —OR, wherein R is independently selected from: substituted or unsubstituted (C1-C18) alkyl, alkenyl, alkynyl and aryl, each inclusive of carbocyclic and heterocyclic. These substituents are readily identified in comparative and competitive enzyme assays.
DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
The following descriptions of particular embodiments and examples are offered by way of illustration and not by way of limitation.
The general method involves forming a mixture of a polymerase and a nucleoside triphosphate (NTP) comprising &agr;, &bgr; and &ggr; phosphates and a &ggr;-phosphate phosphoester-linked functional group; and incubating the mixture under conditions wherein the polymerase catalyzes cleavage of the NTP between the &agr; and &bgr; phosphates, liberating a pyrophosphate comprising the functional group and polymerizing the resultant nucleoside monophosphate. The mixture generally also comprises a template, a nascent polynucleotide and other reagents which facilitate the polymerase reaction, such as salts, buffers, etc. The mixture may be formed in any context, such as in vitro, within a virus or cell, etc. Monitoring polynucleotide synthesis by continuous measurement assays can be performed with homopolymeric templates and a single labeled NTP. Alternatively, continuous monitoring of polymerase activity can be performed by synthesizing all four modified nucleotides, rendering all nucleotides resistant to alkaline phosphatase.
A wide variety of polymerases may be employed, including DNA- and/or RNA-dependent RNA polymerases and DNA- and/or RNA-dependent DNA polymerases. Depending on the application, the polymerase may reside in a cell or virus, such as within its natural host cell environment, or be isolated or in vitro, such as isolated from cellular, microbial and/or viral source material. In many cases, suitable polymerases are commercially available, e.g. Taq, a DNA-dependent DNA polymerase (Boehringer Mannheim (BM) catalog #1-146-165); Klenow fragment (BM catalog #1-008-404) reverse transcriptase (RT), e.g. Moloney murine leukemia virus RT (BM catalog #1-062-603), human immunodeficiency virus RT (BM catalog #1-465-333). Exemplary targetable pathogenic polymerases include reverse transcriptases (e.g. from HIV, and hepatitis B), viral RNA polymerases (e.g. from HCV and Dengues virus) and DNA polymerases (e.g. Herpes and Epstein-Barr virus DNA polymerases).
A wide variety of NTPs which function as substrates for the targeted polymerase may be used in the method. The nucleotide may comprise a conventional purine or pyrimidine base, such as adenine, guanine, cytosine, uracil and thymine, which may be substituted with a variety of known modifications, such as methyl, amine, halide (e.g. 5-fluorouracil), etc., and a pentose (including ribose and deoxyribose), which may also be substituted with a variety of known modifications, such as amine, o-methyl ester, 2′-deoxy, etc. The nucleotide may also comprise a nucleotide analog which functions as a substrate of the target polymerase, such as acyclovir, gangcyclovir, zidovudine (AZT), etc.
The NTP comprises one or two &ggr;-phosphate phosphoester-linked functional groups (i.e. mono phosphoester or phosphodiesters), providing a functionality such as enhancement of reaction product detectability, cell delivery, polymerase specificity, etc. and/or a reaction product functionality such as a therapeutic or protherapeutic, which functionalities may be provided by a wide variety of structural moieties. Numerous exemplary suitable functional groups are disclosed herein and/or readily identified in convenient screens, such as the polymerase, targeting and specificity screens described below.
In one embodiment, the functional group is a detectable label such as a chromogenic or luminogenic (including fluorogenic) label. In this embodiment, the method generally further comprises, after the incubating step, the step of detecting the label. Accordingly, this aspect of the invention provides safe, simple, efficient, nonradioactive, quantitative assays to detect nucleic acid (RNA and DNA) synthesis by polymerases. The methods need not require a separation step where the substrate does not absorb at the detection wavelength until after it is used in a polymerase reaction. Detection can be effected with conventional spectrophoto/fluori-meters routinely used in research laboratories and classrooms. The methods
Epp Jeffrey
Kao C. Cheng
Vassiliou William
Widlanski Theodore
Advanced Research and Technology Institute, Inc.
Osman Richard Aron
Riley Jezia
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