Analysis of the RNA backbone: structural analysis of riboswitches by in-line probing and selective 2'-hydroxyl acylation and primer extension.

RNA sequences fold upon themselves to form complex structures. Functional analysis of most biological RNAs requires knowledge of secondary structure arrangements and tertiary base interactions. Therefore, rapid and comprehensive methods for assessing RNA structure are highly desirable. Computational tools are oftentimes employed for prediction of secondary structure. However, a greater degree of accuracy is achieved when these methods are combined alongside structure probing experimentation. Multiple probing techniques have been developed to assist identification of base-paired regions. However, most of these techniques investigate only a subset of RNA nucleotides at a time. A combination of structure probing approaches is thus required for analysis of all nucleotides within a given RNA molecule. Therefore, methods that investigate local structure for all positions in a sequence-independent manner can be particularly useful in characterizing secondary structure and RNA conformational changes. This chapter outlines protocols for two techniques, in-line probing and Selective 2'-Hydroxyl Acylation Analyzed by Primer Extension (SHAPE), which largely accomplish this goal.