In-gel probing of individual RNA conformers within a mixed population reveals a dimerization structural switch in the HIV-1 leader.
Definitive secondary structural mapping of RNAs in vitro can be complicated by the presence of more than one structural conformer or multimerization of some of the molecules. Until now, probing a single structure of conformationally flexible RNA molecules has typically relied on introducing stabilizing mutations or adjusting buffer conditions or RNA concentration. Here, we present an in-gel SHAPE (selective 2'OH acylation analysed by primer extension) approach, where a mixed structural population of RNA molecules is separated by non-denaturing gel electrophoresis and the conformers are individually probed within the gel matrix. Validation of the technique using a well-characterized RNA stem-loop structure, the HIV-1 trans-activation response element, showed that authentic structure was maintained and that the method was accurate and highly reproducible. To further demonstrate the utility of in-gel SHAPE, we separated and examined monomeric and dimeric species of the HIV-1 packaging signal RNA. Extensive differences in acylation sensitivity were seen between monomer and dimer. The results support a recently proposed structural switch model of RNA genomic dimerization and packaging, and demonstrate the discriminatory power of in-gel SHAPE.
Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge, Cambridgeshire, CB2 0QQ, UK and HIV-Drug Resistance Program, Centre for Cancer Research, National Cancer Institute, P.O. Box B, Building 535, Frederick, MD 21702-1201, USA.
Julia C
JC
Kenyon
Liam J
LJ
Prestwood
Stuart F J
SF
Le Grice
Andrew M L
AM
Lever
D020121:5' Untranslated Regions; D000215:Acylation; D001483:Base Sequence; D019281:Dimerization; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics*; D008958:Models, Molecular; D015335:Molecular Probes; D008969:Molecular Sequence Data; D063347:Native Polyacrylamide Gel Electrophoresis; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000737:chemistry*
D020121:5' Untranslated Regions; D015335:Molecular Probes; D012367:RNA, Viral