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Preparation of messenger rna transcripts for secondary structure analysis using sp 6 polymerase guanylyltransferase and preparative gel electrophoresis



Preparation of messenger rna transcripts for secondary structure analysis using sp 6 polymerase guanylyltransferase and preparative gel electrophoresis



Gene Analysis Techniques 3(3): 45-52



Undegraded radiolabeled messenger RNA (mRNA) is required for the experimental analysis of mRNA secondary structure. The introduction of plasmids containing the SP6 promoter allows the in vitro synthesis of relatively large quantities of purified mRNA for biochemical analysis, and these primary transcripts may be radioactively labeled in the 5' cap structure (m7GpppG) by incubation with .alpha.-[32P]-GTP, vaccinia guanylyltransferase, and S-adenosylmethionine. Because the enzyme requires a 5' di- or tri-phosphate terminus as a substrate, internal degradation products of the RNA are not labeled; moreover, the capped mRNAs appear to be more stable than uncapped. After a final purification by electrophoresis through a preparative polyacrylamide gel, the cap-labeled mRNA preparations are very highly enriched in full-length molecules, which serve as excellent substrates for partial digestion with structure-specific nucleases or chemicals. The relative ease of preparation of cap-labeled RNAs will facilitate analysis of translation-level control mechanisms including mRNA structure and mRNA-protein interactions.

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