5'-sulfhydryl-modified RNA: initiator synthesis, in vitro transcription, and enzymatic incorporation.
The detailed syntheses of the sulfhydryl-modified guanosine monophosphates 5'-deoxy-5'-thioguanosine-5'-monophosphorothioate (GSMP), O-[omega-sulfhydryl-tetra(ethylene glycol)]-O-(5'-guanosine) monophosphate (5'-HS-PEG4-GMP), and O-[omega-sulfhydryl-di(ethylene glycol)]-O-(5'-guanosine) monophosphate (5'-HS-PEG2-GMP) are reported. Transcription reactions employing GSMP, 5'-HS-PEG4-GMP, or 5'-HS-PEG2-GMP as the initiator nucleotide for T7 RNA polymerase introduce a thiol group at the 5'-end of RNA. The efficiency of thiol incorporation at the 5'-terminus of modified RNA compounds was assayed with three different thiol-reactive biotinylated reagents followed by streptavidin gel-shift methods. The transcription efficiency with various ratios of GTP to 5'-HS-PEG2-GMP was explored by reaction with a sulfhydryl-reactive maleimide-conjugated protein. This is an efficient method to incorporate enzymatically a thiol group into the 5'-end of RNA.
Zhang
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Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
Sun
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Cui
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Gottlieb
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RL
Zhang
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B
D019921:Biotinylation; D002474:Cell-Free System; D018387:Codon, Initiator; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D006151:Guanosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry; D012313:RNA / Q000032:analysis / Q000096:biosynthesis*; D013873:Thionucleotides / Q000032:analysis / Q000138:chemical synthesis / Q000378:metabolism*; D014158:Transcription, Genetic
C432004:5'-deoxy-5'-thioguanosine-5'-monophosphorothioate; D018387:Codon, Initiator; D013873:Thionucleotides; D006151:Guanosine; D011092:Polyethylene Glycols; D012313:RNA; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase