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

Abstract

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.