Abstract
In recent years, single-molecule fluorescence resonance energy transfer (smFRET) has emerged as a powerful technique to study macromolecular interactions. The chief advantages of smFRET analysis compared to bulk measurements include the possibility to detect sample heterogeneities within a large population of molecules and the facility to measure kinetics without needing the synchronization of intermediate states. As such, the methodology is particularly well adapted to observe and analyze RNA/RNA and RNA/protein interactions involved in small noncoding RNA-mediated gene regulation networks. In this chapter, we describe and discuss protocols that can be used to measure the dynamics of these interactions, with a particular emphasis on the advantages—and experimental pitfalls—of using the smFRET methodology to study sRNA-based biological systems.
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Abbreviations
- smFRET:
-
Single-molecule Förster resonance energy transfer
- sRNA:
-
Small noncoding RNA
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Acknowledgments
This work was supported by the CNRS, CEA, ESPCI, and University Paris Diderot. Ulrich Bockelmann acknowledges support by the Human Frontier Science Program [RGP008/2014]. We are particularly grateful to Emmanuel Margeat and Erwin Peterman for help with setup design and fruitful discussions, to Rahul Roy, Sungchul Hohng, and Wonseok Hwang for many fruitful discussions and to Jingyi Fei for critical reading of the manuscript.
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Bizebard, T., Arluison, V., Bockelmann, U. (2018). Single-Molecule FRET Assay to Observe the Activity of Proteins Involved in RNA/RNA Annealing. In: Arluison, V., Valverde, C. (eds) Bacterial Regulatory RNA. Methods in Molecular Biology, vol 1737. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7634-8_17
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DOI: https://doi.org/10.1007/978-1-4939-7634-8_17
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