An RNase P-Based Assay for Accurate Determination of the 5'-Deoxy-5'-azidoguanosine-Modified Fraction of in Vitro-Transcribed RNAs.

Chemoenzymatic approaches are important for generating site-specific, chemically modified RNAs, a cornerstone for RNA structure-function correlation studies. T7 RNA polymerase (T7RNAP)-mediated in vitro transcription (IVT) of a DNA template containing the G-initiating class III Φ6.5 promoter is typically used to generate 5'-chemically modified RNAs by including a guanosine analogue (G analogue) initiator in the IVT. However, the yield of 5'-G analogue-initiated RNA is often poor and variable due to the high ratios of G analogue:GTP used in IVT. We recently reported that a T7RNAP P266L mutant afforded an approximately three-fold increase in fluorescent 5'-thienoguanosine-initiated pre-tRNACys compared to the wild type T7RNAP. We have further explored the use of T7RNAP P266L to generate 5'-deoxy-5'-azidoguanosine (az G)-initiated RNA and found that the mutant yielded approximately four times more az G-initiated pre-tRNACys than the wild type in an IVT containing a 10:1 ratio of az G:GTP. For accurate quantitation of the 5'-az G-initiated RNA fraction, we employed RNase P, an endonuclease that catalyzes the removal of the 5'-leader in pre-tRNAs. Importantly, we show herein how RNase P can be leveraged for assessing 5'-G analogue incorporation in any RNA by rendering the target RNA, upon its binding to a customized external guide sequence RNA, into an unnatural substrate of RNase P. Such an approach in conjunction with T7RNAP P266L-based IVT should aid chemoenzymatic methods that are designed to generate 5'-chemically modified RNAs.