Efficient synthesis of nucleic acids heavily modified with non-canonical ribose 2'-groups using a mutantT7 RNA polymerase (RNAP).
A T7 RNAP mutant (Y639F) which eliminates discrimination of the chemical character of the NTP ribose 2'-group, facilitates incorporation of non-canonicalsubstrates into nucleic acids. However, transcripts containing a high percentage of non-canonical NMPs are poorly extended due to effects of the 2'-substituents on the transcript:template hybrid conformation. We tested the addition of compounds that stabilize A-type helix geometry to the reaction. High concentrations of polyamines, together with other changes in reaction conditions, greatly increased the synthesis of transcripts heavily substituted with non-canonical ribose 2'-groups. Template structures that facilitate promoter opening increased the efficiency of reactions where non-canonical substrates were incorporated during transcription of +1 to +6.
Padilla
R
R
Department of Biochemistry, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio,TX 78284-7760, USA.
Sousa
R
R
D017123:Bacteriophage T7 / Q000201:enzymology*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009696:Nucleic Acids / Q000096:biosynthesis*; D012265:Ribonucleotides / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D014158:Transcription, Genetic; D014764:Viral Proteins
D009696:Nucleic Acids; D012265:Ribonucleotides; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases