Chemical modification interference.

Abstract

Chemical modification interference is a powerful method for surveying an entire RNA molecule to identify functionally important chemical groups. The basic idea is to generate a pool of end-labeled RNAs wherein each RNA molecule is chemically modified (e.g., by diethyl pyrocarbonate [DEPC], hydrazine, dimethyl sulfate, CMCT, or kethoxal) at a different position. The pool of RNAs is then allowed to participate in the reaction of interest. The functionally important RNA molecules (e.g., those bound by protein or that successfully participate in a processing reaction) are then separated from the nonfunctional RNA molecules (e.g., those not bound by protein or unable to participate in a processing reaction). This is often achieved by straightforward gel electrophoretic analysis. In the case of protein binding, it is necessary to be able to separate bound RNA from unbound RNA, which can be accomplished using electrophoretic mobility shift assays, filter binding, or affinity approaches (e.g., by immunoprecipitation or the use of tagged proteins). None of these techniques requires that a large fraction of RNA be bound or reacted, and, as a result, they are quite sensitive. Here we describe one example of a chemical modification interference assay in which RNA is modified with DEPC or hydrazine before binding to a protein. This technique can be readily adapted for use with other chemicals.