A Y639F/H784A T7 RNA polymerase double mutant displays superior properties for synthesizing RNAs with non-canonical NTPs.
A T7 RNA polymerase in which Tyr639 is mutated to Phe readily utilizes 2'-deoxy, 2'-NH2 and 2'-F NTPs as substrates and has been widely used to synthesize modified RNAs for a variety of applications. This mutant does not readily utilize NTPs with bulkier 2'-substituents, nor does it facilitate incorporation of NTPs with modifications at other positions. Introduction of a second mutation (H784A) into the Y639F background markedly enhances utilization of NTPs with bulky 2'-substituents (2'-OMe and 2'-N3), and may also enhance use of NTPs with modifications at other than the 2'-position. The Y639F/H784A double mutant may therefore be exceptionally useful for incorporation of a variety of non-canonical NMPs into RNA.
Padilla
Robert
R
Department of Biochemistry, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA.
Sousa
Rui
R
D019943:Amino Acid Substitution; D001386:Azides / Q000737:chemistry; D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D012313:RNA / Q000096:biosynthesis / Q000235:genetics*; D012265:Ribonucleotides / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D014158:Transcription, Genetic; D014764:Viral Proteins
D001386:Azides; D012265:Ribonucleotides; D014764:Viral Proteins; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases