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Single Molecule Studies of RNA–RNA Interactions

  • Dongmei Yu
  • Peiwu Qin
  • Peter V. CornishEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1240)

Abstract

Non-coding RNAs including microRNAs, siRNAs, and snoRNAs interact with their targets directly through RNA–RNA interactions by base-paring (van Himbergen et al., Nucleic Acids Res 21(8):1713–1717, 1993). RNA–RNA interactions play important roles in gene transcription and protein translation, which can be investigated with several experimental techniques including single molecule methods. Here, we describe how single molecule Förster resonance energy transfer (FRET) can be used to study RNA–RNA interactions in vitro by either surface immobilization or vesicle encapsulation.

Key words

RNA–RNA interactions RNA ligation RNA labeling Single molecule FRET 

Notes

Acknowledgements

This work was supported by an NSF CAREER award (MCB-115343). P.C. is a Pew Scholar in the Biomedical Sciences.

References

  1. 1.
    Zhuang X (2005) Single-molecule RNA science. Annu Rev Biophys Biomol Struct 34:399–414PubMedCrossRefGoogle Scholar
  2. 2.
    Roy R, Hohng S, Ha T (2008) A practical guide to single-molecule FRET. Nat Methods 5(6):507–516PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Nilsen TW, Rio DC (2012) In vitro transcription of labeled RNA: synthesis, capping, and substitution. Cold Spring Harb Protoc 2012(11):1181–1186PubMedGoogle Scholar
  4. 4.
    Cazenave C, Uhlenbeck OC (1994) RNA template-directed RNA synthesis by T7 RNA polymerase. Proc Natl Acad Sci U S A 91(15):6972–6976PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Richardson RW, Gumport RI (1983) Biotin and fluorescent labeling of RNA using T4 RNA ligase. Nucleic Acids Res 11(18):6167–6184PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Kinoshita Y, Nishigaki K, Husimi Y (1997) Fluorescence-, isotope- or biotin-labeling of the 5'-end of single-stranded DNA/RNA using T4 RNA ligase. Nucleic Acids Res 25(18):3747–3748PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Solomatin S, Herschlag D (2009) Methods of site-specific labeling of RNA with fluorescent dyes. Methods Enzymol 469:47–68PubMedCrossRefGoogle Scholar
  8. 8.
    Joo C, Ha T (2012) Labeling DNA (or RNA) for single-molecule FRET. Cold Spring Harb Protoc 2012(9):1005–1008PubMedGoogle Scholar
  9. 9.
    Joo C, Ha T (2012) Preparing sample chambers for single-molecule FRET. Cold Spring Harb Protoc 2012(10):1104–1108PubMedGoogle Scholar
  10. 10.
    Diao J et al (2012) A single vesicle-vesicle fusion assay for in vitro studies of SNAREs and accessory proteins. Nat Protoc 7(5):921–934PubMedCrossRefGoogle Scholar
  11. 11.
    Ha T (2001) Single-molecule fluorescence resonance energy transfer. Methods 25(1):78–86PubMedCrossRefGoogle Scholar
  12. 12.
    McKinney SA, Joo C, Ha T (2006) Analysis of single-molecule FRET trajectories using hidden Markov modeling. Biophys J 91(5):1941–1951PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of BiochemistryUniversity of MissouriColumbiaUSA

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