RNA Chaperones pp 271-282 | Cite as

Single-Molecule FRET Assay for Studying Cotranscriptional RNA Folding

  • Heesoo Uhm
  • Sungchul HohngEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2106)


Cotranscriptional RNA folding plays important roles in gene regulation steps such as splicing, transcription termination, and translation initiation. Progression of our understanding of cotranscriptional RNA folding mechanisms is still retarded by the lacking of experimental tools to study the kinetics of cotranscriptional RNA folding properly. In this chapter, we describe fluorescence resonance energy transfer (FRET) assay that enables the study of RNA cotranscriptional folding at the single-molecule level.

Key words

Cotranscriptional folding Single-molecule FRET Elongation complex reconstitution Riboswitch 



This work was supported by a Creative Research Initiative program (2009-0081562) to SH.


  1. 1.
    Badelt S, Hammer S, Flamm C, Hofacker IL (2015) Chapter 8–thermodynamic and kinetic folding of riboswitches. In: Chen S-J, Burke-Aguero DH (eds) Methods in enzymology. Academic Press: Cambridge, Massachusetts, US, pp 193–213.
  2. 2.
    Draper DE (2008) RNA folding: thermodynamic and molecular descriptions of the roles of ions. Biophys J 95:5489–5495. Scholar
  3. 3.
    Bhaskaran H, Russell R (2007) Kinetic redistribution of native and misfolded RNAs by a DEAD-box chaperone. Nature 449:1014–1018. Scholar
  4. 4.
    Neuman KC, Abbondanzieri EA, Landick R et al (2003) Ubiquitous transcriptional pausing is independent of RNA polymerase backtracking. Cell 115:437–447. Scholar
  5. 5.
    Pan T, Sosnick T (2006) Rna folding during transcription. Annu Rev Biophys Biomol Struct 35:161–175. Scholar
  6. 6.
    Anthony PC, Perez CF, García-García C, Block SM (2012) Folding energy landscape of the thiamine pyrophosphate riboswitch aptamer. Proc Natl Acad Sci 109:1485–1489. Scholar
  7. 7.
    Uhm H, Bae S, Lee M, Hohng S (2016) Single-molecule FRET combined with magnetic tweezers at low force regime. Bull Kor Chem Soc 37:408–410. Scholar
  8. 8.
    Uhm H, Hohng S (2017) Ligand recognition mechanism of thiamine pyrophosphate riboswitch aptamer. Bull Kor Chem Soc 38:1465–1473. Scholar
  9. 9.
    Uhm H, Kang W, Ha KS et al (2018) Single-molecule FRET studies on the cotranscriptional folding of a thiamine pyrophosphate riboswitch. Proc Natl Acad Sci 115:331–336. Scholar
  10. 10.
    Guizar-Sicairos M, Thurman ST, Fienup JR (2008) Efficient subpixel image registration algorithms. Opt Lett 33:156–158. Scholar
  11. 11.
    Hwang W, Bae S, Hohng S (2012) Autofocusing system based on optical astigmatism analysis of single-molecule images. Opt Express 20:29353–29360. Scholar
  12. 12.
    Roy R, Hohng S, Ha T (2008) A practical guide to single-molecule FRET. Nat Methods 5:507–516. Scholar
  13. 13.
    Hohng S, Lee S, Lee J, Jo MH (2014) Maximizing information content of single-molecule FRET experiments: multi-color FRET and FRET combined with force or torque. Chem Soc Rev 43:1007–1013. Scholar
  14. 14.
    Kapanidis AN, Lee NK, Laurence TA et al (2004) Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules. Proc Natl Acad Sci 101:8936–8941. Scholar
  15. 15.
    Daube SS, von Hippel P (1992) Functional transcription elongation complexes from synthetic RNA-DNA bubble duplexes. Science 258:1320–1324. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Physics and AstronomySeoul National UniversitySeoulRepublic of Korea
  2. 2.Institute of Applied PhysicsSeoul National UniversitySeoulRepublic of Korea

Personalised recommendations