Intrinsic properties of RNA to form Watson-Crick base pairing allows it to self-assemble into specific and programmable nano-sized complexes. However, the naturally occurring RNA strands are not stable, and they hydrolyze quickly in blood serum. This limits RNA application, for example, as a nanovehicle for targeted drug delivery. The replacement of the hydroxyl group at the 2’ position of the ribose to 2’-Fluoro (2’-F) or 2’-Methoxy (2’-Met) can drastically elevate the resistance of the RNA to nucleases and improve overall stability. However, to synthesize such modified RNAs, a mutated version of traditionally used T7 RNA polymerase is often required. The recombinant RNApol will not discriminate between regular riboNucleotideTriphosphates (rNTPs) vs modified-rNTPs and, hence, can be implemented to transcribe modified RNA strands. Herein, we describe overexpression and isolation of RGVG and Y639F RNA polymerases from E.Coli cells using metal-ion immobilized affinity chromatography. We demonstrated that in optimized conditions, these RNA polymerases can be used to obtain milligram quantities of 2’-F and 2’-Met RNA polymers possessing high levels of resistance to nuclease degradation in blood serum.

Overexpression and isolation of RGVG and Y639F mutants of T7 RNA polymerase for <i>in vitro</i> transcription of modified RNA strands

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Overexpression and isolation of RGVG and Y639F mutants of T7 RNA polymerase for in vitro transcription of modified RNA strands