Transcription yield of fully 2'-modified RNA can be increased by the addition of thermostabilizing mutations to T7 RNA polymerase mutants

Nucleic Acids Res. 2015 Sep 3;43(15):7480-8. doi: 10.1093/nar/gkv734. Epub 2015 Jul 24.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA-Directed RNA Polymerases / chemistry
  • DNA-Directed RNA Polymerases / genetics*
  • DNA-Directed RNA Polymerases / metabolism
  • Enzyme Stability / genetics
  • Mutation*
  • RNA / biosynthesis
  • RNA / chemistry
  • Substrate Specificity
  • Temperature
  • Transcription, Genetic*
  • Viral Proteins / chemistry
  • Viral Proteins / genetics*
  • Viral Proteins / metabolism


  • Viral Proteins
  • RNA
  • bacteriophage T7 RNA polymerase
  • DNA-Directed RNA Polymerases