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Novel Biochemical Tools for Probing HIV RNA Structure

  • Protocol
HIV Protocols

Part of the book series: Methods in Molecular Biology ((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.

Jason W. Rausch and Joanna Sztuba-Solinska have equally contributed to this chapter.

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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

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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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Author notes

    Authors and Affiliations

    1. Reverse Transcriptase Biochemistry Section, HIV Drug Resistance Program, Frederick National Laboratory for Cancer Research, Frederick, MD, 21702, USA

      Jason W. Rausch & Joanna Sztuba-Solinska

    2. Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892, USA

      Sabrina Lusvarghi

    3. Reverse Transcriptase Biochemistry Section, HIV Drug Resistance Program, Frederick National Laboratory for Cancer Research, Building 535, Room 312, Frederick, MD, 21702, USA

      Stuart F. J. Le Grice

    Authors
    1. Jason W. Rausch
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    2. Joanna Sztuba-Solinska
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    3. Sabrina Lusvarghi
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    4. Stuart F. J. Le Grice
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    Corresponding author

    Correspondence to Stuart F. J. Le Grice .

    Editor information

    Editors and Affiliations

    1. Department of Microbiology & Immunology, Albert Einstein College of Medicine, BRONX, New York, USA

      Vinayaka R. Prasad

    2. Department of Genetics, Albert Einstein College of Medicine, BRONX, New York, USA

      Ganjam V. Kalpana

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    Rausch, J.W., Sztuba-Solinska, J., Lusvarghi, S., Le Grice, S.F.J. (2016). Novel Biochemical Tools for Probing HIV RNA Structure. In: Prasad, V., Kalpana, G. (eds) HIV Protocols. Methods in Molecular Biology, vol 1354. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3046-3_7

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    • DOI: https://doi.org/10.1007/978-1-4939-3046-3_7

    • Publisher Name: Humana Press, New York, NY

    • Print ISBN: 978-1-4939-3045-6

    • Online ISBN: 978-1-4939-3046-3

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