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 Φ6.5 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 ( G):GTP increased the percent incorporation and yield of 5'- 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'- G-modified transcripts. This approach should be of broad utility in preparing 5'-modified RNAs.

An RNase P‐Based Assay for Accurate Determination of the 5′‐Deoxy‐5′‐azidoguanosine‐Modified Fraction of in Vitro‐Transcribed RNAs

“…[4] We recently demonstrated [4] that the decreased yield could be partly overcome by use of the Pro266Leu (P266L) mutant derivative of T7RNAP, which has a decreased propensity for abortive transcription. [5] Use of the P266L mutant compared to the wild type (WT) [4] afforded a 3-fold increase in the IVT yield of pre-tRNA Cys initiated with thienoguanosine ( th G), a fluorescent guanosine surrogate.…”

“…[4] We recently demonstrated [4] that the decreased yield could be partly overcome by use of the Pro266Leu (P266L) mutant derivative of T7RNAP, which has a decreased propensity for abortive transcription. [5] Use of the P266L mutant compared to the wild type (WT) [4] afforded a 3-fold increase in the IVT yield of pre-tRNA Cys initiated with thienoguanosine ( th G), a fluorescent guanosine surrogate. [6] When an IVT performed with this mutant was coupled with a one-pot multi-enzyme (OPME) method (Figure S1), [4] which entails a post-transcriptional clean-up step using sequentially a pyrophosphohydrolase and an exoribonuclease to specifically degrade 5’-GTP-initiated pre-tRNA Cys , we obtained near-homogeneous 5’- th G-initiated pre-tRNA Cys at good yield (40 μg/100 μL IVT).…”

“…In our recent study, [4] we chose to study 5’- th G-initiated RNAs because the intrinsic fluorescence of th G together with Abs 260 measurements permitted an easy quantitation of the fraction of in vitro transcribed RNAs initiated with either th G or GTP. While exploring the potential of T7RNAP P266L to synthesize RNAs initiated with non-fluorescent G-analogs, we recognized the need for an accurate method to assess the fractional content of GTP- versus G-analog-initiated RNAs present at the end of an IVT.…”

“…For pairwise comparisons, we used ~2 μg of T7RNAP WT and P266L in our IVTs as described previously. [4] First, we examined if aborted transcription with T7RNAP WT was indeed a roadblock in IVTs containing az G. For a sensitive visualization in this regard, we conducted “hot”, small-scale transcriptions with α− 32 P-UTP. With T7RNAP WT, we observed a substantial decrease in the full-length (likely due to an increase in aborted transcripts), when the az G:GTP ratio was increased progressively from 0:1 to 4:1 to 10:1 (Figure S2).…”

“…In contrast, T7RNAP P266L produced more full-length pre-tRNA Cys than T7RNAP WT regardless of the az G:GTP ratio used (Figure S2); this finding mirrors our previous observation with th G as the G-analog initiator in IVTs. [4] …”