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Nucleic Acids Res. 1999 Mar 15;27(6):1561-3.

Efficient synthesis of nucleic acids heavily modified with non-canonical ribose 2'-groups using a mutantT7 RNA polymerase (RNAP).

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1
Department of Biochemistry, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio,TX 78284-7760, USA.

Abstract

A T7 RNAP mutant (Y639F) which eliminates discrimination of the chemical character of the NTP ribose 2'-group, facilitates incorporation of non-canonicalsubstrates 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.

PMID:
10037823
PMCID:
PMC148355
[Indexed for MEDLINE]
Free PMC Article
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