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Identification of a T7 RNA polymerase variant that permits the enzymatic synthesis of fully 2′-O-methyl-modified RNA

Paper ID Volume ID Publish Year Pages File Format Full-Text
23508 43444 2013 9 PDF Available
Title
Identification of a T7 RNA polymerase variant that permits the enzymatic synthesis of fully 2′-O-methyl-modified RNA
Abstract

•A single amino acid substitution converts wildtype T7 RNA polymerase into a processive 2′-O-methyl nucleotide polymerase.•Residue R425 is located at the end of a sequence motif (DX2GR) which is conserved among many DNA and RNA polymerases.•An all-2′-O-methyl-modified aptamer analog retains function by binding the cognate ligand of its natural antetype.

T7 RNA polymerase is an important biocatalyst that is used in diverse biotechnological applications such as in vitro transcription or protein expression. The enzyme displays high substrate specificity which is payed by significant limitations regarding incorporation of synthetic nucleotide analogs. Of specific interest is enzymatic synthesis of 2′-O-methyl-modified RNA as these nucleic acids exhibit improved biochemical and pharmacokinetic properties that make them attractive for diagnostic and therapeutic purposes. We report here on the development of an activity-based selection/screening approach for assessing polymerase activities in the presence of 2′-O-methyl-modified nucleotides, and on the identification of one variant T7 RNA polymerase which is capable of synthesizing all-2′-O-methyl RNA.

Keywords
RNA; Polymerase; Modified nucleotide; Aptamer
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Identification of a T7 RNA polymerase variant that permits the enzymatic synthesis of fully 2′-O-methyl-modified RNA
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 167, Issue 3, 10 September 2013, Pages 287–295
Authors
, , , , , ,
Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
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Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
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