Plasmid Template Design and In Vitro Transcription of Short RNAs Within a “Structure Cassette” for Structure Probing Experiments

Abstract

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.

Dissection of the Adenoviral VA RNAI Central Domain Structure Reveals Minimum Requirements for RNA-mediated Inhibition of PKR

“…All new RNAs prepared here contain the A2dl2 mutation, but for brevity they are named only for their terminal deletion or point mutation(s) made within the TS⌬21-A2dl2 context. New templates were created by direct ligation of 5Ј end phosphorylated double-stranded DNA into a plasmid created previously for the transcription of target RNAs with a 3Ј end-fused hepatitis delta virus (3Ј-HDV) ribozyme (24,25). RNAs were produced by runoff in vitro transcription using T7 RNA polymerase under optimal conditions for VA RNA I (26), purified by preparative denaturing PAGE, and recovered as described previously (8,27).…”

“…0.5, 10, 30, and 240 min at 85, 50, 40, and 20°C, respectively (28). Reverse transcription was performed with Superscript III reverse transcriptase (Invitrogen) using a 5Ј end ␥ 32 P-labeled DNA primer (0.12 M final concentration) corresponding to the sequence of the 3Ј flanking region of the structure cassette (25) in a total volume of 10 l in 100 mM HEPES buffer (pH 8.0) containing 100 mM NaCl. Reactions were stopped by addition of 35 l of a 4:25 (v/v) mixture of 1 M unbuffered Tris-HCl and gel loading solution (85% formamide, 0.5ϫ TBE (pH 8.0) and 50 mM EDTA with trace bromphenol blue and xylene cyanol dyes) and heating to 95°C for 5 min.…”

“…The A123U construct was made exclusively with the NaeI runoff site. RNAs for SHAPE and Tb 3ϩ probing were generated by the same direct ligation approach but using a modified plasmid preloaded with 5Ј and 3Ј flanking sequences encoding a "structure cassette" (25,28).…”

SHAPE analysis of the htrA RNA thermometer from Salmonella enterica

“…The structure was predicted using mfold 3.0 (Zuker 2003), as shown in Klinkert et al (2012). (B) The putative structure of the htrA thermometer embedded in the SHAPE structure cassette (Wilkinson et al 2006;Vachon and Conn 2012). The primer-binding site is shown in blue, and the remainder of the cassette regions are shown in gray.…”

“…For this, the entire 40-nt 5 ′ UTR plus the first two codons of the coding region were embedded in a set of stable hairpins known as a structure cassette ( Fig. 1B; Wilkinson et al 2006;Vachon and Conn 2012). The structure cassette provides a binding site for primer extension and prevents information loss from the 5 ′ and 3 ′ ends of the RNA of interest, allowing all nucleotides of the RNA thermometer to be simultaneously assessed.…”