Structural analysis of RNA using chemical and enzymatic probing monitored by primer extension.

Publisher Summary This chapter focuses on the structural analysis of RNA using chemical and enzymatic probing monitored by primer extension. Chemical and enzymatic probing, monitored by primer extension, has become a powerful tool for the analysis of RNA structure. The reactivities of individual nucleotides composing large RNA molecules may be determined rapidly by utilizing a series of primers spaced at approximately 200 nucleotide intervals. In addition, numerous chemical reagents and nucleases may be employed as probes, since the only requirement is that they modify the template so as to produce pauses or stops in the progress of reverse transcriptase. The RNA, either alone or complexed with proteins and/or ligands, is incubated under suitable conditions with chemical or enzymatic probes. Dimethyl sulfate (DMS), kethoxal (KE) and l-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho- p -toluene sulfonate (CMCT) are employed for chemical probing, while ribonucleases A and T 1 , and V l nuclease are employed as enzymatic probes. The extent of the reactions is limited so that no more than a few stops are present within 300 nucleotide stretches in a given RNA molecule.