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

  • Somdeb MitraEmail author
  • Borries Demeler
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2113)

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.

Key words

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

Notes

Acknowledgments

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.

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Authors and Affiliations

  1. 1.Department of ChemistryNew York UniversityNew YorkUSA
  2. 2.Department of Chemistry and BiochemistryUniversity of LethbridgeLethbridgeCanada

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