A mutation in T7 RNA polymerase enhances the yield of 5'-guanosine-analog-initiated RNAs
Loading...
Date
2018-05
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
The Ohio State University
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 nucleotides (nt), the enzymatic incorporation of a nucleoside or nucleoside monophosphate guanosine analog (G-analog) 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 Φ6.5 promoter. However, when high G-analog:GTP ratios are used to bias G-analog incorporation at the 5'-end, RNA yield is compromised. Here, we show that the use of a T7RNAP Pro266Leu mutant in an IVT with 10:1 thienoguanosine (thG, a fluorescent guanosine surrogate):GTP increased the percent incorporation and yield of 5'-thG-initiated precursor tRNA for a net three-fold gain compared to an IVT with wild-type T7RNAP. We also demonstrate that a one-pot multi-enzyme (OPME) approach, consisting of transcription by T7RNAP Pro266Leu and post-transcriptional cleanup by a polyphosphatase and an exonuclease, led to essentially near-homogeneous 5'-thG-modified transcripts. We also validated the broader utility of our one-pot multi-enzyme approach to initiate RNAs with an azide-bearing G-analog, an undertaking that necessitated the development an RNase P (a tRNA 5′-maturation endonuclease)-based assay to accurately determine the percentage of 5'-azide-initiated RNA. The OPME approach, coupled with use of T7RNAP Pro266Leu, should be of broad utility in generating near homogenous 5'-G-analog RNAs.
Description
Keywords
Fluorophores, T7 RNA Polymerase, RNA modification, in vitro transcription