Nucleoside phosphorylase polynucleotide synthesis

The first enzyme discovered that could catalyze polynucleotide synthesis was a bacterial enzyme called polynucleotide phosphorylase. This enzyme, isolated by Severo Ochoa and Marianne Grunberg-Manago in 1955, could make long chains of 5 -3 -linked polyribonucleotides starting from nucleoside diphosphates. However, there was no template requirement for this synthesis, and the sequence was uncontrollable except in a crude way by adjusting the relative concentrations of different nucleotides in the starting materials. 

Adenine is inert in the nucleoside phosphorylase systems of both mammalian tissues and microorganisms, but isotopically labeled adenine is effectively incorporated into nucleic acid purines, both in rats " and in yeast.This poses a question as to the possible role of nucleoside phosphorylase in polynucleotide synthesis. It has been suggested that hypoxanthine or guanine nucleosides (or nucleotides) are synthesized first. Then an exchange reaction with free adenine (or a derivative) might occur, For example, adenine might react with inosine to form adenosine and hypoxanthine. Some known exchange reactions are discussed below. 

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. ... 

When chemical and enzymatic methods for NTP synthesis are compared[941, enzymatic techniques provide the most convenient route to CTP and GTP, whereas chemical deamination of CTP is the best method for preparing UTP 94L ATP is relatively inexpensive from commercial sources, although it has been synthesized enzymatically from AMP on 50 mmol scale. Mixtures of NTPs can be prepared from RNA by sequential nuclease Pi, polynucleotide phosphorylase, and pyruvate kinase-catalyzed reactions11101. This mixture can be selectively converted to a sugar nucleotide using a particular sugar nucleoside diphosphate pyrophosphorylase11101. 

Polynucleotide phosphorylase an enzyme catalysing the synthesis in vitro of polyribonucleotides. 5 -Nucleoside diphosphates are added to oligonucleotide starter molecules (primers) with the release of phosphate. The resulting sequence depends on the availability of components for the reaction. Thus it is possible to synthesize homopolymers (poly A, poly U, etc.) or Copolymers (see) (e.g. poly AU). The function of P.p. in the cell is not clear. Since it also acts as an exonuclease and catalyses the reverse reaction, it is possible that the enzyme functions in the synthesis and remobilization of storage polynucleotides. 

The kinase step enables cells to utilize nucleosides from their milieu for the incorporation into nucleotide coenzymes and polynucleotides, and has the obvious value that synthesis de novo is spared. The kinase reaction, operating in sequence with the readily reversible uridine phosphorylase reaction, also provides a route by which uracil may enter into the pyrimidine nucleotide pool. 

Reaction conditions have been described for the synthesis of oligonucleotides by the use of polynucleotide phosphorylase, which uses a dinucleoside (3 -50-nionophosphate as primer and nucleoside 5 -phos-phates. The enzyme also used as primers unnatural phosphodiesters, such as nitrophenylated nucleoside 5 -phosphates. The yields of these reactions, however, are considerably lower as compared to reactions with natural phosphodiesters as primer. The yields of the reaction were found to be relatively insensitive to variations in salt concentration or in pH of the incubation mixtures. Optimal salt conditions, i.e., mM MgCh and 0.4-1,0 MNaCl, are similar to those that are used in oligonucleotide synthesis with normal primers. However, it is advisable that incubation mixtures not exceed volumes of 1 ml. In our experience, it is better to split larger volumes into smaller ones, if large quantities of oligonucleotide are to be synthesized. 

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