title: Transcription yield of fully 2'-modified RNA can be increased by the addition of thermostabilizing mutations to T7 RNA polymerase mutants.
abstract: On average, mutations are deleterious to proteins. Mutations conferring new function to a protein often come at the expense of protein folding or stability, reducing overall activity. Over the years, a panel of T7 RNA polymerases have been designed or evolved to accept nucleotides with modified ribose moieties. These modified RNAs have proven useful, especially in vivo, but the transcriptional yields tend to be quite low. Here we show that mutations previously shown to increase the thermal tolerance of T7 RNA polymerase can increase the activity of mutants with expanded substrate range. The resulting polymerase mutants can be used to generate 2'-O-methyl modified RNA with yields much higher than enzymes currently employed.
authors.affiliation: Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA andy.ellington@mail.utexas.edu.
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Meyer
authors.affiliation: Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Garry
authors.affiliation: Altermune Technologies, LLC, Irvine, CA 92606, USA.
authors.forename: Bradley
authors.initials: B
authors.lastname: Hall
authors.affiliation: Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA.
authors.forename: Michelle M
authors.initials: MM
authors.lastname: Byrom
authors.affiliation: Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA.
authors.forename: Hannah G
authors.initials: HG
authors.lastname: McDonald
authors.affiliation: Department of Pharmacology and Toxicology, Sealy Center for Structural Biology, University of Texas Medical Branch, Galveston, TX 77555, USA.
authors.forename: Xu
authors.initials: X
authors.lastname: Yang
authors.affiliation: Department of Pharmacology and Toxicology, Sealy Center for Structural Biology, University of Texas Medical Branch, Galveston, TX 77555, USA.
authors.forename: Y Whitney
authors.initials: YW
authors.lastname: Yin
authors.affiliation: Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, TX 78712, USA Center for Systems & Synthetic Biology, University of Texas at Austin, Austin, TX 78712, USA.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Ellington
mesh_terms: D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability / Q000235:genetics; D009154:Mutation; D012313:RNA / Q000096:biosynthesis / Q000737:chemistry; D013379:Substrate Specificity; D013696:Temperature; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D014764:Viral Proteins; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases