title: Synthesis of 2'-Fluoro RNA by Syn5 RNA polymerase.
abstract: 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.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA Bin_Zhu@hms.harvard.edu.
authors.forename: Bin
authors.initials: B
authors.lastname: Zhu
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Alfredo
authors.initials: A
authors.lastname: Hernandez
authors.affiliation: Departments of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Min
authors.initials: M
authors.lastname: Tan
authors.affiliation: Institute of Chemistry and Biochemistry, Free University Berlin, Berlin 14195, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Wollenhaupt
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Stanley
authors.initials: S
authors.lastname: Tabor
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Charles C
authors.initials: CC
authors.lastname: Richardson
mesh_terms: D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism; D005461:Fluorine / Q000737:chemistry; D008345:Manganese; D009154:Mutation; D017902:Podoviridae / Q000201:enzymology; D012313:RNA / Q000096:biosynthesis* / Q000737:chemistry; D020871:RNA Stability; D024363:Transcription Initiation Site; D014158:Transcription, Genetic
chemical_list: D003854:Deoxyribonucleotides; D005461:Fluorine; D008345:Manganese; D012313:RNA; D012321:DNA-Directed RNA Polymerases