Efficient synthesis of nucleic acids heavily modified with non-canonical ribose 2'-groups using a mutant T7 RNA polymerase (RNAP)

Robert Padilla, Rui Sousa

Research output: Contribution to journalArticlepeer-review

105 Scopus citations

Abstract

A T7 RNAP mutant (Y639F) which eliminates discrimination of the chemical character of the NTP ribose 2'-group, facilitates incorporation of non-canonical substrates into nucleic acids. However, transcripts containing a high percentage of non-canonical NMPs are poorly extended due to effects of the 2'-substituents on the transcript:template hybrid conformation. We tested the addition of compounds that stabilize A-type helix geometry to the reaction. High concentrations of polyamines, together with other changes in reaction conditions, greatly increased the synthesis of transcripts heavily substituted with non-canonical ribose 2'-groups. Template structures that facilitate promoter opening increased the efficiency of reactions where non-canonical substrates were incorporated during transcription of +1 to +6.

Original languageEnglish (US)
Pages (from-to)1561-1563
Number of pages3
JournalNucleic acids research
Volume27
Issue number6
DOIs
StatePublished - Mar 15 1999

ASJC Scopus subject areas

  • Genetics

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