A Y639F/H784A T7 RNA polymerase double mutant displays superior properties for synthesizing RNAs with non-canonical NTPs.

A T7 RNA polymerase in which Tyr639 is mutated to Phe readily utilizes 2'-deoxy, 2'-NH2 and 2'-F NTPs as substrates and has been widely used to synthesize modified RNAs for a variety of applications. This mutant does not readily utilize NTPs with bulkier 2'-substituents, nor does it facilitate incorporation of NTPs with modifications at other positions. Introduction of a second mutation (H784A) into the Y639F background markedly enhances utilization of NTPs with bulky 2'-substituents (2'-OMe and 2'-N3), and may also enhance use of NTPs with modifications at other than the 2'-position. The Y639F/H784A double mutant may therefore be exceptionally useful for incorporation of a variety of non-canonical NMPs into RNA.