Zhu, BinHernandez, AlfredoTan, MinWollenhaupt, JanTabor, StanleyRichardson, Charles2015-09-012015Zhu, Bin, Alfredo Hernandez, Min Tan, Jan Wollenhaupt, Stanley Tabor, and Charles C. Richardson. 2015. “Synthesis of 2′-Fluoro RNA by Syn5 RNA polymerase.” Nucleic Acids Research 43 (14): e94. doi:10.1093/nar/gkv367. http://dx.doi.org/10.1093/nar/gkv367.0305-1048http://nrs.harvard.edu/urn-3:HUL.InstRepos:21462414The 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.en-USJournal Article2015-09-0110.1093/nar/gkv367