Evolution of a T7 RNA polymerase variant that transcribes 2'-O-methyl RNA.
Modified RNA and DNA molecules have novel properties that their natural counterparts do not possess, such as better resistance to degradation in cells and improved pharmacokinetic behavior. In particular, modifications at the 2'-OH of ribose are important for enhancing the stability of RNA. Unfortunately, it is difficult to enzymatically synthesize modified nucleic acids of any substantial length because natural polymerases incorporate modified nucleotides inefficiently. Previously, we reported an activity-based method for selecting functional T7 RNA polymerase variants based on the ability of a T7 RNA polymerase to reproduce itself. Here, we have modified the original procedure to identify polymerases that can efficiently incorporate multiple modified nucleotides at the 2' position of the ribose. Most important, our method allows the selection of polymerases that have good processivities and can be combined to simultaneously incorporate several different modified nucleotides in a transcript.
Jijumon
J
Chelliserrykattil
Andrew D
AD
Ellington
D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism*; D019020:Directed Molecular Evolution / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D014644:Genetic Variation; D008745:Methylation; D015202:Protein Engineering / Q000379:methods*; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics*; D014764:Viral Proteins
D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases