Plasmid template design and in vitro transcription of short RNAs within a "structure cassette" for structure probing experiments.

Chemical and enzymatic RNA structure probing methods are important tools for examining RNA secondary and tertiary structures and their interactions with proteins, small molecules, and ions. The recently developed "Selective 2'-Hydroxyl Acylation Analyzed by Primer Extension" (SHAPE) approach has proven especially useful for uncovering details of secondary structures, complex tertiary interactions, and RNA dynamics. Analysis of short RNAs using SHAPE or other probing methods that require reverse transcription to detect RNA modifications presents a technical hurdle in that intense bands corresponding to abortive transcription during primer extension and the full-length RT product may obscure information corresponding to the 3' and 5' ends of the molecule, respectively. This chapter describes the design and use of an RNA "structure cassette" that addresses these issues. First, we describe methods by which any RNA of interest may be cloned into a new plasmid preloaded with sequences that encode a structure cassette surrounding the short internal target RNA. Next, we outline key considerations and analyses of the RNAs produced that should be performed prior to SHAPE or other structure probing experiments.