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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Zhuang X (2005) Single-molecule RNA science. Annu Rev Biophys Biomol Struct 34:399–414
Roy R, Hohng S, Ha T (2008) A practical guide to single-molecule FRET. Nat Methods 5(6):507–516
Nilsen TW, Rio DC (2012) In vitro transcription of labeled RNA: synthesis, capping, and substitution. Cold Spring Harb Protoc 2012(11):1181–1186
Cazenave C, Uhlenbeck OC (1994) RNA template-directed RNA synthesis by T7 RNA polymerase. Proc Natl Acad Sci U S A 91(15):6972–6976
Richardson RW, Gumport RI (1983) Biotin and fluorescent labeling of RNA using T4 RNA ligase. Nucleic Acids Res 11(18):6167–6184
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):3747–3748
Solomatin S, Herschlag D (2009) Methods of site-specific labeling of RNA with fluorescent dyes. Methods Enzymol 469:47–68
Joo C, Ha T (2012) Labeling DNA (or RNA) for single-molecule FRET. Cold Spring Harb Protoc 2012(9):1005–1008
Joo C, Ha T (2012) Preparing sample chambers for single-molecule FRET. Cold Spring Harb Protoc 2012(10):1104–1108
Diao J et al (2012) A single vesicle-vesicle fusion assay for in vitro studies of SNAREs and accessory proteins. Nat Protoc 7(5):921–934
Ha T (2001) Single-molecule fluorescence resonance energy transfer. Methods 25(1):78–86
McKinney SA, Joo C, Ha T (2006) Analysis of single-molecule FRET trajectories using hidden Markov modeling. Biophys J 91(5):1941–1951
Acknowledgements
This work was supported by an NSF CAREER award (MCB-115343). P.C. is a Pew Scholar in the Biomedical Sciences.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Yu, D., Qin, P., Cornish, P.V. (2015). Single Molecule Studies of RNA–RNA Interactions. In: Schmidt, F. (eds) RNA-RNA Interactions. Methods in Molecular Biology, vol 1240. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1896-6_7
Download citation
DOI: https://doi.org/10.1007/978-1-4939-1896-6_7
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-1895-9
Online ISBN: 978-1-4939-1896-6
eBook Packages: Springer Protocols