Probing RNA–Protein Interactions and RNA Compaction by Sedimentation Velocity Analytical Ultracentrifugation

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Abstract

Recent advances in multi-wavelength analytical ultracentrifugation (MWL-AUC) combine the power of an exquisitely sensitive hydrodynamic-based separation technique with the added dimension of spectral separation. This added dimension has opened up new doors to much improved characterization of multiple, interacting species in solution. When applied to structural investigations of RNA, MWL-AUC can precisely report on the hydrodynamic radius and the overall shape of an RNA molecule by enabling precise measurements of its sedimentation and diffusion coefficients and identify the stoichiometry of interacting components based on spectral decomposition. Information provided in this chapter will allow an investigator to design experiments for probing ion and/or protein-induced global conformational changes of an RNA molecule and exploit spectral differences between proteins and RNA to characterize their interactions in a physiological solution environment.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages281-317
Number of pages37
DOIs
StatePublished - 2020

Publication series

NameMethods in Molecular Biology
Volume2113
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Funding

S.M. is grateful to the Chemistry Department of New York University to host him as a faculty during the preparation of this manuscript. The Twort intron work described here was originally funded by 1RO1-GM085130 from the National Institute of General Medical Sciences of the National Institutes of Health to Prof. Michael D. Brenowitz at the Albert Einstein College of Medicine. B.D. wishes to credit NIH-NIGMS grant RO1-120600 and the Canada Research Chairs program for financial support of this work.

FundersFunder number
1RO1-GM085130
RO1-120600
R01GM085130
Albert Einstein College of Medicine

    Keywords

    • Counterion
    • Hydrodynamic measurements
    • Multi-wavelength analytical ultracentrifugation
    • RNA folding
    • Sedimentation velocity

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