A simple and efficient method to reduce nontemplated nucleotide addition at the 3 terminus of RNAs transcribed by T7 RNA polymerase.
DNA templates modified with C2'-methoxyls at the last two nucleotides of the 5' termini dramatically reduced nontemplated nucleotide addition by the T7 RNA polymerase from both single- and double-stranded DNA templates. This strategy was used to generate several different transcripts. Two of the transcripts were demonstrated by nuclear magnetic resonance spectroscopy to be unaffected in their sequence. Transcripts produced from the modified templates can be purified with greater ease and should be useful in a number of applications.
Kao
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C
Department of Biology, Indiana University, Bloomington 47405, USA. ckao@bio.indiana.edu
Zheng
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M
Rüdisser
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S
D001483:Base Sequence; D004247:DNA / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D009682:Magnetic Resonance Spectroscopy; D008967:Molecular Biology; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000138:chemical synthesis; D012313:RNA / Q000138:chemical synthesis*; D012266:Ribose / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic; D014764:Viral Proteins
D009841:Oligonucleotides; D014764:Viral Proteins; D012313:RNA; D012266:Ribose; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases