Nucleoside triphosphates, enzymatic

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. ... 

The unique properties of oligonucleotides create crosslinking options that are far different from any other biological molecule. Nucleic acids are the only major class of macromolecule that can be specifically duplicated in vitro by enzymatic means. The addition of modified nucleoside triphosphates to an existing DNA strand by the action of polymerases or transferases allows addition of spacer arms or detection components at random or discrete sites along the chain. Alternatively, chemical methods that modify nucleotides at selected functional groups can be used to produce spacer arm derivatives or activated intermediates for subsequent coupling to other molecules. 

Enzymatic techniques can employ a variety of DNA or RNA polymerases to add controlled amounts of modified nucleotides to an existing stand. However, the most common procedures utilize either DNA polymerase I or terminal deoxynucleotide transferase. The polymerase is used with a template to add modified nucleoside triphosphates to the end of a DNA molecule or to various sites within the middle of a sequence. The terminal transferase can add modified monomers to the 3 end of a chain without a template. 

Regardless of the type of enzymatic labeling used, it is important that the label be incorporated into the nucleoside triphosphates or primers in a way that does not affect enzyme recognition and activity. Thus, every enzymatic labeling procedure for modifying RNA or DNA probes must start with chemical derivatization of individual nucleotides. Of the many chemical procedures that can be used to modify a nucleoside triphosphate monomer, there are only a few that will result in a derivative still able to be enzymatically added to an existing oligonucleotide strand. 

The pyrimidine nucleosides dUTP or dCTP can be modified at their C-5 position with a spacer arm containing a tag, such as a biotin group, and still remain good substrates for DNA polymerase. Enzymatic labeling with a biotin-modified pyrimidine nucleoside triphosphate is one of the most common methods of adding a detectable group to an existing DNA strand. 

Figure 27.1 Three common nucleoside triphosphate derivatives that can be incorporated into oligonucleotides by enzymatic means. The first two are biotin derivatives of pyrimidine and purine bases, respectively, that can be added to an existing DNA strand using either polymerase or terminal transferase enzymes. Modification of DNA with these nucleosides results in a probe detectable with labeled avidin or streptavidin conjugates. The third nucleoside triphosphate derivative contains an amine group that can be added to DNA using terminal transferase. The modified oligonucleotide then can be labeled with amine-reactive bioconjugation reagents to create a detectable probe.

Some form of chemical labeling process must be used regardless of whether the final oligo conjugate is created by enzymatic or strictly chemical means. If enzymatic modification is to be done, the initial label still must be incorporated into an individual nucleoside triphosphate, which then is polymerized into an existing oligonucleotide strand (Section 1, this chapter). Fortunately, many useful modified nucleoside triphosphates are now available from commercial sources, often eliminating the need for custom derivatization of individual nucleotides. 

Radiolabeled RNA can be generated either by incorporation of a (a32p) nucleoside triphosphate during an in vitro transcription reaction or by the transfer of (y P)-ATP to the 5 terminus of a dephosphorylated RNA molecule (41). The authors prefer the first mentioned method, as it needs only a single enzymatic reaction. 

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 ... 

By contrast, derivatization at the ends of an oligo or at the sugar—phosphate backbone usually produces little interference in base-pairing. Conjugates may be created by enzymatic polymerization of functionalized nucleoside triphosphates off the 3 end or... 

Enzymatic labeling using any of these polymerase methods results in derivatized nucleoside triphosphates being incorporated at numerous locations within an oli-... 

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... 

Enzymatic routes to analogues of nucleoside triphosphates have also been described but are not included here.19... 

Fig. 4 Ferrocene-, anthraquinone- and ruthenium-derivitized electrochemical nucleoside triphosphates (electrotides) compatible with enzymatic incorporation into nucleic acids. Examples include a Fcl-UTP (adapted from [184]), b Aql-UTP (adapted from [184]), c Fcl-acyUTP (adapted from [185]), d ferrocene-acycloATP (adapted from [278]), e Fc2-dUTP (adapted from [181]), f Fcl-dUTP (adapted from [181]), g Aql-dCTP (adapted from [185]) and h dRuTP (adapted from [188])...

Most of the enzymatic reactions that utilize the naturally occurring diamagnetic Mg ion still occur when the Mg is substituted by the paramagnetic Mrf ion, ofren with little or no loss of activity. Since Mn is present at much lower concentrations than Mg in the cell, Mn does not play a role in vivo. The substitution studies are nonetheless useful, since Mn can be used as a probe for cation environment. An important class of enzymes that have been studied in this way are those enzymes that utilize nucleoside triphosphates, including elongation factors and kinases. 

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

Nucleotides can also be prepared by using enzymatic reactions. Modified nucleoside triphosphates are useful as precursors for... 

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