Zhu, Bin; Hernandez, Alfredo; Tan, Min; Wollenhaupt, Jan; Tabor, Stanley; Richardson, Charles C.
Year of publication:
The substitution of 2′-fluoro for 2′-hydroxyl moieties in RNA substantially
improves the stability of RNA. RNA stability is a major issue in RNA research
and applications involving RNA. We report that the RNA polymerase from the
marine cyanophage Syn5 has an intrinsic low discrimination against the
incorporation of 2′-fluoro dNMPs during transcription elongation. The presence
of both magnesium and manganese ions at high concentrations further reduce
this discrimination without decreasing the efficiency of incorporation. We
have constructed a Syn5 RNA polymerase in which tyrosine 564 is replaced with
phenylalanine (Y564F) that further decreases the discrimination against 2
′-fluoro-dNTPs during RNA synthesis. Sequence elements in DNA templates that
affect the yield of RNA and incorporation of 2′-fluoro-dNMPs by Syn5 RNA
polymerase have been identified.