A T7 RNA Polymerase Mutant Enhances the Yield of 5'-Thienoguanosine-Initiated RNAs.

ABSTRACT: Spectroscopic methods, which are used to establish RNA structure-function relationships, require strategies for post-synthetic, site-specific incorporation of chemical probes into target RNAs. For RNAs larger than 50?nt, the enzymatic incorporation of a nucleoside or nucleotide monophosphate guanosine analogue (G?analogue) at their 5'-end is routinely achieved by T7 RNA polymerase (T7RNAP)-mediated in vitro transcription (IVT) of the appropriate DNA template containing a GTP-initiating class?III ?6.5 promoter. However, when high G?analogue:GTP ratios are used to bias G?analogue incorporation at the 5'-end, RNA yield is compromised. Here, we show that the use of a T7RNAP P266L mutant in IVT with 10:1 thienoguanosine (th G):GTP increased the percent incorporation and yield of 5'-th G-initiated precursor tRNA for a net ?threefold gain compared to IVT with wild-type T7RNAP. We also demonstrated that a one-pot multienzyme approach, consisting of transcription by T7RNAP P266L and post-transcriptional cleanup by polyphosphatase and an exonuclease, led to essentially near-homogeneous 5'-th G-modified transcripts. This approach should be of broad utility in preparing 5'-modified RNAs.

SUBMITTER: Lyon S 

PROVIDER: S-EPMC6047071 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature