Native Purification and Labeling of RNA for Single Molecule Fluorescence Studies

  • Arlie J. Rinaldi
  • Krishna C. Suddala
  • Nils G. Walter
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1240)

Abstract

The recent discovery that non-coding RNAs are considerably more abundant and serve a much wider range of critical cellular functions than recognized over previous decades of research into molecular biology has sparked a renewed interest in the study of structure–function relationships of RNA. To perform their functions in the cell, RNAs must dominantly adopt their native conformations, avoiding deep, non-productive kinetic traps that may exist along a frustrated (rugged) folding free energy landscape. Intracellularly, RNAs are synthesized by RNA polymerase and fold co-transcriptionally starting from the 5′ end, sometimes with the aid of protein chaperones. By contrast, in the laboratory RNAs are commonly generated by in vitro transcription or chemical synthesis, followed by purification in a manner that includes the use of high concentrations of urea, heat and UV light (for detection), resulting in the denaturation and subsequent refolding of the entire RNA. Recent studies into the nature of heterogeneous RNA populations resulting from this process have underscored the need for non-denaturing (native) purification methods that maintain the co-transcriptional fold of an RNA. Here, we present protocols for the native purification of an RNA after its in vitro transcription and for fluorophore and biotin labeling methods designed to preserve its native conformation for use in single molecule fluorescence resonance energy transfer (smFRET) inquiries into its structure and function. Finally, we present methods for taking smFRET data and for analyzing them, as well as a description of plausible overall preparation schemes for the plethora of non-coding RNAs.

Key words

RNA folding Non-denaturing purification Single molecule fluorescence resonance energy transfer Non-denaturing RNA transcription Fluorophore labeling of RNA Biotin labeling of RNA 

Notes

Acknowledgments

Work in the authors’ laboratory was supported by the NIH grants RO1 GM062357, GM098023, and GM06316.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Arlie J. Rinaldi
    • 4
    • 5
    • 6
  • Krishna C. Suddala
    • 2
    • 3
  • Nils G. Walter
    • 1
    • 2
  1. 1.Department of ChemistryUniversity of MichiganThe Claremont Colleges, ClaremontUSA
  2. 2.BiophysicsUniversity of MichiganAnn ArborUSA
  3. 3.Single Molecule Analysis GroupUniversity of MichiganAnn ArborUSA
  4. 4.W. M. Keck Science CenterThe Claremont CollegesClaremontUSA
  5. 5.Department of ChemistryUniversity of Michigan930 N. University, Ann ArborUSA
  6. 6.Single Molecule Analysis GroupUniversity of MichiganAnn ArborUSA

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