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

Adam J. Meyer, Daniel J. Garry, Bradley Hall, Michelle M. Byrom, Hannah G. McDonald, Xu Yang, Yuhui Yin, Andrew D. Ellington

Research output: Contribution to journalArticle

19 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)7480-7488
Number of pages9
JournalNucleic Acids Research
Volume43
Issue number15
DOIs
StatePublished - Jul 7 2015
Externally publishedYes

Fingerprint

RNA
Mutation
Ribose
Protein Stability
Protein Folding
Proteins
Nucleotides
Enzymes
bacteriophage T7 RNA polymerase
Thermotolerance

ASJC Scopus subject areas

  • Genetics

Cite this

Meyer, A. J., Garry, D. J., Hall, B., Byrom, M. M., McDonald, H. G., Yang, X., ... Ellington, A. D. (2015). Transcription yield of fully 2'-modified RNA can be increased by the addition of thermostabilizing mutations to T7 RNA polymerase mutants. Nucleic Acids Research, 43(15), 7480-7488. https://doi.org/10.1093/nar/gkv734

Transcription yield of fully 2'-modified RNA can be increased by the addition of thermostabilizing mutations to T7 RNA polymerase mutants. / Meyer, Adam J.; Garry, Daniel J.; Hall, Bradley; Byrom, Michelle M.; McDonald, Hannah G.; Yang, Xu; Yin, Yuhui; Ellington, Andrew D.

In: Nucleic Acids Research, Vol. 43, No. 15, 07.07.2015, p. 7480-7488.

Research output: Contribution to journalArticle

Meyer, Adam J. ; Garry, Daniel J. ; Hall, Bradley ; Byrom, Michelle M. ; McDonald, Hannah G. ; Yang, Xu ; Yin, Yuhui ; Ellington, Andrew D. / Transcription yield of fully 2'-modified RNA can be increased by the addition of thermostabilizing mutations to T7 RNA polymerase mutants. In: Nucleic Acids Research. 2015 ; Vol. 43, No. 15. pp. 7480-7488.
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