Transcription yield of fully 2′-modified RNA can be increased by the addition of thermostabilizing mutations to T7 RNA polymerase mutants

Abstract

On average, mutations are deleterious to proteins. Mutations conferring new function to a protein often come at the expense of protein folding or stability, reducing overall activity. Over the years, a panel of T7 RNA polymerases have been designed or evolved to accept nucleotides with modified ribose moieties. These modified RNAs have proven useful, especially in vivo, but the transcriptional yields tend to be quite low. Here we show that mutations previously shown to increase the thermal tolerance of T7 RNA polymerase can increase the activity of mutants with expanded substrate range. The resulting polymerase mutants can be used to generate 2′-O-methyl modified RNA with yields much higher than enzymes currently employed.

From selection hits to clinical leads: progress in aptamer discovery

“…Most recently, improved variants of the Chelliserrykattil and Ellington polymerase variant RGVG 82 were described by Meyer et al . 84 By including a series of stabilizing mutations into the enzyme, they were able to further increase the yields of 100% 2’OMe RNA transcriptions to levels approaching those observed for the wild-type polymerases using unmodified nucleotides. 84 …”

The expanding world of DNA and RNA

“…Optimized conditions and the use of the Y639F/H784A double mutant of T7 RNAP has also enabled the transcription of oligonucleotides with increased levels of 2’-OMe substitutions, which have been used for the identification of stabilized aptamers against vascular endothelial growth factor (VEGF) [5] and staphylococcal protein A [6]. The recent discovery of additional T7 RNAP mutants that better recognize 2’-OMe substrates [7,8] and a mutant of the Syn5 RNA polymerase from cyanophage Syn5 that better recognizes 2’-F substrates [9] should facilitate the identification of stabilized aptamers in the future.…”

A T7 RNA Polymerase Mutant Enhances the Yield of 5′‐Thienoguanosine‐Initiated RNAs

“…First, we explored the use of a mutant T7RNAP containing 10 mutations (termed ‘RGVG-M6’), including P266L, which exhibited a 25-fold increase in RNA yield of fully 2'-modified RNA over other commonly used mutant T7RNAPs. [7d] When we purified this mutant and tested the recombinant version in IVTs, we did not observe an increase in the total yield of 5'- th G-initiated RNAs. Second, we examined the effects of retaining the [ th G]:[GTP] ratio but increasing their absolute concentrations on T7RNAP P266L’s activity.…”

“…[6] From this panel of mutants, we chose T7RNAP Pro266Leu (P266L) because kinetic and footprinting studies revealed that this mutant exhibits a delayed transition from transcriptional initiation to elongation and generates fewer aborted transcripts. [7] We sought to test the postulate that the T7RNAP P266L mutant would decrease aborted transcripts typically observed when 5'-G-analogs are used with T7RNAP wild-type (WT; Figure 2A ). The problem of low G-analog incorporation, however, necessitates a combination of transcriptional and post-transcriptional strategies since a fraction of transcripts will always be initiated with GTP.…”

“…The findings with th G reaffirm the increased tolerance of T7RNAP P266L mutant to initiate RNA transcripts with modified nucleosides, an attribute noted before while investigating use of modified 2'-NTPs. [7c] Given the value of th G as a probe of RNA folding/dynamics and RNA function, [5b] the facile synthesis of 5'- th G-initiated RNAs should increase the use of this guanosine isomorph. Moreover, we expect that the OPME approach described here will improve percent incorporation and yields of other G-analogs at the 5'-end of RNAs of any length and sequence.…”