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Stepwise synthesis of oligo nucleotides 29. poly nucleotide phosphorylase in oligo ribo nucleotide synthesis



Stepwise synthesis of oligo nucleotides 29. poly nucleotide phosphorylase in oligo ribo nucleotide synthesis



Bioorganicheskaya Khimiya 6(10): 1505-1515



Micrococcus luteus polynucleotide phosphorylase can be effectively used for the synthesis of triribonucleoside diphosphates containing 3'-terminal pyrimidine nucleotide. The enzyme catalyzes the addition of only 1 nucleotide residue to dinucleoside monophosphate with maximal yield, provided the initial concentrations of a nucleoside 5'-diphosphate and a dinucleoside monophosphate acceptor are 5-10 mM and 10-20 mM, respectively, substrates are incubated with the enzyme at 37.degree. C no more than 1 h for ppU and 2 h for ppC. The temperature rise to 50.degree. C results in the increase of the phosphate acceptor consumption due to formation of longer oligonucleotides and their following phosphorolysis. The fall of the temperature decreases considerably the trinucleoside diphosphate yield, whereas the amount of longer oligonucleotides is not reduced. The trinucleoside diphosphate yield depends on the structures of nucleoside 5'-diphosphate and of the phosphate acceptor. The structural requirements of the enzyme for more active addition of pyrimidine nucleotide residues are not altered at higher temperature. The optimal conditions may be found for each pair of substrates. Polynucleotide phosphorylase from E. coli, similarly to the M. luteus enzyme, catalyzes the addition of only 1 nucleotide residue to a dinucleoside monophosphate, the pyrimidine nucleotides being attached more effectively. The enzyme concentration necessary for the effective addition depends on the phosphate acceptor length.

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