Selective 5' modification of T7 RNA polymerase transcripts.
We have developed two methods for selective 5' modification of RNAs generated by enzymatic synthesis using T7 RNA polymerase. The first method involves a two-step procedure. Transcription reactions are performed under standard conditions except that GTP is replaced by GTP gamma S. Since the polymerase initiates transcription with GTP, every transcript contains a 5' gamma-thiophosphate group which is modified with the thiol-specific reagent of choice (e.g., iodoacetyl dansyl derivative) in the second step. In an alternative method, transcription and modification reactions are carried out in a single step, using a mixture of dansylated GTP and GTP. Under the appropriate conditions, dansylated GTP effectively competes with GTP in the initiation reaction but does not substantially inhibit the elongation reaction. Yields of fluorescent 64-mer RNA ranging from 30 to 70% of the total transcription product have been obtained using these methods in combination with HPLC purification. This approach is amenable to large scale synthesis reactions and can be used to produce a wide variety of 5'-modified RNAs of virtually any size for structural or functional studies.
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Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
Lee
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Harper
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D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000737:chemistry; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins
D012333:RNA, Messenger; D014764:Viral Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases