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.