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Novel Biochemical Tools for Probing HIV RNA Structure

  • Jason W. Rausch
  • Joanna Sztuba-Solinska
  • Sabrina Lusvarghi
  • Stuart F. J. Le Grice
Part of the Methods in Molecular Biology book series (MIMB, volume 1354)

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.

Key words

Ensemble SHAPE In-gel SHAPE Methidiumpropyl-EDTA ATCUN Chemical probing HIV RNA 

Abbreviations

ATCUN

Amino terminal copper- and nickel-binding motif

MPE

Methidiumpropyl-EDTA

ND

Non-denaturing

PAGE

Polyacrylamide gel electrophoresis

RRE

Rev response element

SHAPE

Selective 2′-hydroxyl acylation analyzed by primer extension

TAR

Trans-activation response element

Notes

Acknowledgements

This work was funded by the Intramural Research Program of the National Cancer Institute, National Institutes of Health, Department of Health and Human Services. The authors would like to thank Jennifer Miller for critical reading of the manuscript.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Jason W. Rausch
    • 1
  • Joanna Sztuba-Solinska
    • 1
  • Sabrina Lusvarghi
    • 2
  • Stuart F. J. Le Grice
    • 3
  1. 1.Reverse Transcriptase Biochemistry Section, HIV Drug Resistance ProgramFrederick National Laboratory for Cancer ResearchFrederickUSA
  2. 2.Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney DiseasesNational Institutes of HealthBethesdaUSA
  3. 3.Reverse Transcriptase Biochemistry Section, HIV Drug Resistance ProgramFrederick National Laboratory for Cancer ResearchFrederickUSA

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