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

Robert Padilla | Zendy

Open Access

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

Author(s) -

Robert Padilla

Publication year - 1999

Publication title -

nucleic acids research

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 9.008

H-Index - 537

eISSN - 1362-4954

pISSN - 0305-1048

DOI - 10.1093/nar/27.6.1561

Subject(s) - biology , nucleic acid , ribose , transcription (linguistics) , polymerase , t7 rna polymerase , dna , rna , non canonical , rna polymerase , mutant , biochemistry , microbiology and biotechnology , genetics , stereochemistry , biophysics , enzyme , gene , chemistry , bacteriophage , escherichia coli , philosophy , linguistics

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.

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