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A T7 RNA Polymerase Mutant Enhances the Yield of 5′‐Thienoguanosine‐Initiated RNAs
Author(s) -
Lyon Seth,
Gopalan Venkat
Publication year - 2018
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201700538
Subject(s) - t7 rna polymerase , rna , transcription (linguistics) , gtp' , guanosine , exonuclease , rna polymerase iii , polymerase , nucleotide , mutant , biology , rna polymerase , biochemistry , dna , transfer rna , chemistry , microbiology and biotechnology , enzyme , gene , escherichia coli , linguistics , philosophy , bacteriophage
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 ( 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.