SYNTHESIS OF MIXED RIBO DEOXYRIBOPOLYNUCLEOTIDES BY MUTANT T7 RNA-POLYMERASE/

Synthesis of deoxynucleotide-containing RNA-like single-stranded polyn ucleotides (dcRNAs) using the Y639F, S611A mutant of T7 RNA polymerase (T7 RNAP) was studied. A number of different T7 promoter-containing p lasmids were tested as templates for dcRNA synthesis, The dcRNA synthe sis efficiency strongly depended on the sequence of the first 8-10 nuc leotides immediately downstream of the promoter and increased with the distance of the first incorporated dNMP from the transcription start. The incorporation of dGMP which is obligatory for most T7 promoters i n positions +1-+2(3) was practically negligible. Using the constructed plasmid pTZR7G containing seven dG links in the non-coding chain imme diately downstream of the promoter, the synthesis of all possible dcRN As (except dG-containing) was achieved with high yields, (C) 1998 Fede ration of European Biochemical Societies.