title: Novel Biochemical Tools for Probing HIV RNA Structure.
abstract: Functional analysis of viral RNA requires knowledge of secondary structure arrangements and tertiary base interactions. Thus, high-throughput and comprehensive methods for assessing RNA structure are highly desirable. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) has proven highly useful for modeling the secondary structures of HIV and other retroviral RNAs in recent years. This technology is not without its limitations however, as SHAPE data can be severely compromised when the RNA under study is structurally heterogeneous. In addition, the method reveals little information regarding the three-dimensional (3D) organization of an RNA. This chapter outlines four detailed SHAPE-related methodologies that circumvent these limitations. "Ensemble" and "in-gel" variations of SHAPE permit structural analysis of individual conformers within structurally heterogeneous mixtures of RNA, while probing strategies that utilize "through-space" cleavage reagents such as methidiumpropyl-EDTA (MPE) and peptides appended with an ATCUN (amino terminal copper/nickel binding motif) can provide insight into 3D organization. Combinational application of these techniques provides a formidable arsenal for exploring the structures of HIV RNAs and associated nucleoprotein complexes.
authors.affiliation: Reverse Transcriptase Biochemistry Section, HIV Drug Resistance Program, Frederick National Laboratory for Cancer Research, Frederick, MD, 21702, USA.
authors.forename: Jason W
authors.initials: JW
authors.lastname: Rausch
authors.affiliation: Reverse Transcriptase Biochemistry Section, HIV Drug Resistance Program, Frederick National Laboratory for Cancer Research, Frederick, MD, 21702, USA.
authors.forename: Joanna
authors.initials: J
authors.lastname: Sztuba-Solinska
authors.affiliation: Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892, USA.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Lusvarghi
authors.affiliation: Reverse Transcriptase Biochemistry Section, HIV Drug Resistance Program, Frederick National Laboratory for Cancer Research, Building 535, Room 312, Frederick, MD, 21702, USA. legrices@mail.nih.gov.
authors.forename: Stuart F J
authors.initials: SF
authors.lastname: Le Grice
mesh_terms: D001665:Binding Sites; D019075:Electrophoresis, Capillary / Q000379:methods; D006678:HIV / Q000737:chemistry* / Q000378:metabolism; D015658:HIV Infections / Q000821:virology; D006801:Humans; D008958:Models, Molecular; D063347:Native Polyacrylamide Gel Electrophoresis / Q000379:methods; D009690:Nucleic Acid Conformation; D010455:Peptides / Q000378:metabolism; D059370:RNA Folding; D015347:RNA Probes / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012367:RNA, Viral / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D048348:Reverse Transcription
chemical_list: D010455:Peptides; D015347:RNA Probes; D012367:RNA, Viral
keywords: ATCUN; Chemical probing; Ensemble SHAPE; HIV RNA; In-gel SHAPE; Methidiumpropyl-EDTA