Nucleoside triphosphates, enzymatic syntheses

C. Oligo- and Poly-nucleotides.—The stepwise enzymatic synthesis of internucleotide bonds has been reviewed. A number of polynucleotides containing modified bases have been synthesised " in the past year from nucleoside triphosphates with the aid of a polymerase enzyme, and the enzymatic synthesis of oligodeoxyribonucleotides using terminal deoxynucleotidyl transferase has been studied. Primer-independent polynucleotide phosphorylase from Micrococcus luteus has been attached to cellulose after the latter has been activated with cyanogen bromide. The preparation of insolubilized enzyme has enabled large quantities of synthetic polynucleotides to be made. The soluble enzyme has been used to prepare various modified polycytidylic acids. ... 

Several chemical and enzymatic methods are available for the synthesis of glycosyl phosphates. The required nucleoside triphosphates (NTPs) are most conveniently prepared by enzymatic routes. In general, these methods involve the sequential use of two kinases to transform NMPs to NTPs, by the corresponding NDPs (Fig. 2, see a recent review [12] for more details). 

Before the sequencing begins it is necessary to prepare a short primer that is complementary to a sequence at one end of the DNA strand to be sequenced. This may be prepared enzymatically,622 623 or by non-enzymatic synthesis. The short primer is annealed to the end of the DNA and the resulting molecule is incubated with a DNA polymerase and a mixture of the four mononucleotide triphosphates, one of which is radio-labeled in this position. Four reaction mixtures are prepared. Each mixture contains all four nucleoside triphosphates and also one of four different chain-terminating inhibitors, the most popular of which are the 2, 3 -dideoxyribonucleoside triphosphates ... 

Polyphosphate kinase has been found able to phosphorylate nucleoside diphosphates to give nucleoside triphosphates, using PolyP as a phosphate donor. Therefore, the possibility of using PolyP and polyphosphate kinase instead of phosphoenol pyruvate and pyruvate kinase for enzymatic oligosaccharide synthesis was examined, because PolyP is quite cheap when compared with phosphoenol pyruvate (Noguchi and Shiba, 1998 Shiba et al, 2000). Attempts were made to synthesize N-acetyllactosamine (Gal (f> 1-4) GlcNAc) using the nucleoside diphosphate kinase-like activity of polyphosphate kinase, where UDP-Glc pyrophosphorylase and UDP-Glc 4-epimerase catalyse the synthesis of UDP-Glc from... 

Sequencing by synthesis Templeted enzymatic incorpora- tion F attached to bases of nucleoside triphosphates Fluorescence sensing, multicolour detection 43... 

Different types of antitemplates can be and, presumably, will be, synthesized by employing other chemical reactions that are sufficiently mild and selective for the modification of polynucleotides. Another approach to this problem is, of course, to synthesize an appropriately modified nucleoside triphosphate and utilize it as one of the substrates in a DNA or RNA polymerizing system, in the presence of the specific template for which an antitemplate is to be made. The enzymatic synthesis method has some obvious advantages however, the inhibitory activity of the product (antitemplate) may limit its applications. 

Preparative-scale synthesis of nucleoside triphosphates. Nucleoside triphosphates are utilized as substrates for the biosynthesis of sugar nucleoside phosphates. Practical-scale biosynthesis-based enzymatic preparation of NTPs for use in glycosylations is therefore required. 

H. Cahova, L. Havran, P. Brazdilova, H. Pivonkova, R. Pohl, M. Fojta, and M. Hocek, Aminophenyl- and nitrophenyl-labeled nucleoside triphosphates Synthesis, enzymatic incorporation, and electrochemical detection, Angew. Chem., hit Ed. 47[11], 2059-2062 [2008]. 

Polymerase enzymes are used extensively in the laboratory for the synthesis and manipulation of DNA and RNA (44). One of the most important applications of enzymatic DNA synthesis is in the polymerase chain reaction (PGR), which is used to amplify minute quantities of DNA from a variety of soin-ces, inclnd-ing crime scene evidence, archaeological specimens, or medical samples (45). In PGR, two short oligonucleotide primers are first synthesized by chemical methods. These primers are complementary to the 5 -ends of the two strands which form the DNA duplex of interest (Fig. 11). A large excess of the primers is mixed with the target DNA along with a DNA polymerase enzyme and the nucleoside triphosphate monomers. The sample is heated to disrupt the target duplex. Then, as the... 

Among the potential targets for VSV inhibition of cellular RNA synthesis are transport of nucleoside triphosphates across the cell membrane, enzymatic conversion to nucleotides, initiation/chain elongation/termination of nuclear chromatin transcription, polyaden-ylation, processing, transport to the cytoplasm of completed RNA transcripts, and stability. Quite obviously, RNA synthesis inhibition could be at the level of ribosomal, messenger, and/or transfer RNA catalyzed by polymerases I, II, and III, respectively. 

We have discussed modified nucleoside phosphoramidites (Section 2.1) and modified nucleoside triphosphates (Section 5.1) and their uses as the monomers for chemical or enzymatic synthesis of modified oligomers (oligonucleotides and oligodeojqmucleotides). In this section, our emphasis is primarily on interesting properties of modified oligomers. 

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