Probing Single Helicase Dynamics on Long Nucleic Acids Through Fluorescence-Force Measurement

Part of the Methods in Molecular Biology book series (MIMB, volume 1486)


Helicases are nucleic acid-dependent ATPases which can bind and remodel nucleic acids, protein–nucleic acid complexes, or both. They are involved in almost every step in cells related to nucleic acid metabolisms, including DNA replication and repair, transcription, RNA maturation and splicing, and nuclear export processes. Using single-molecule fluorescence-force spectroscopy, we have previously directly observed helicase translocation on long single-stranded DNA and revealed that two monomers of UvrD helicase are required for the initiation of unwinding function. Here, we present the details of fluorescence-force spectroscopy instrumentation, calibration, and activity assays in detail for observing the biochemical activities of helicases in real time and revealing how mechanical forces are involved in protein–nucleic acid interaction. These single-molecule approaches are generally applicable to many other protein–nucleic acid systems.

Key words

Single-molecule TIRF Optical tweezers Fluorescence localization Helicase UvrD NS3 Translocation Unwinding 



T. H. is an Investigator of the Howard Hughes Medical Institute. This work is supported by NIH grant GM065367 and NSF grants PHY-1430124 to T. H. We would like to thank Olivia Yang for proofreading the manuscript and Dr. Kyung Suk Lee for constructing the original instrument and training.


  1. 1.
    Lu HP, Xun L, Xie XS (1998) Single-molecule enzymatic dynamics. Science 282(5395):1877–1882. doi: 10.1126/science.282.5395.1877 CrossRefGoogle Scholar
  2. 2.
    Robison AD, Finkelstein IJ (2014) High-throughput single-molecule studies of protein–DNA interactions. FEBS Lett 588(19):3539–3546. doi: 10.1016/j.febslet.2014.05.021 CrossRefGoogle Scholar
  3. 3.
    Ilya JF, Eric CG (2013) Molecular traffic jams on DNA. Annu Rev Biophys 42(1):241–263. doi: 10.1146/annurev-biophys-083012-130304 CrossRefGoogle Scholar
  4. 4.
    Ha T, Enderle T, Ogletree DF et al (1996) Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor. Proc Natl Acad Sci U S A 93(13):6264–6268CrossRefGoogle Scholar
  5. 5.
    Roy R, Hohng S, Ha T (2008) A practical guide to single molecule FRET. Nat Methods 5(6):507–516. doi: 10.1038/nmeth.1208 CrossRefGoogle Scholar
  6. 6.
    Visscher K, Gross SP, Block SM (1996) Construction of multiple-beam optical traps with nanometer-resolution position sensing. IEEE J Sel Top Quantum Electron 2(4):1066–1076. doi: 10.1109/2944.577338 CrossRefGoogle Scholar
  7. 7.
    Comstock MJ, Whitley KD, Jia H et al (2015) Direct observation of structure-function relationship in a nucleic acid–processing enzyme. Science 348(6232):352–354. doi: 10.1126/science.aaa0130 CrossRefGoogle Scholar
  8. 8.
    Moffitt JR, Chemla YR, Izhaky D et al (2006) Differential detection of dual traps improves the spatial resolution of optical tweezers. Proc Natl Acad Sci U S A 103(24):9006–9011. doi: 10.1073/pnas.0603342103 CrossRefGoogle Scholar
  9. 9.
    Woodside MT, Anthony PC, Behnke-Parks WM et al (2006) Direct measurement of the full, sequence-dependent folding landscape of a nucleic acid. Science 314(5801):1001–1004. doi: 10.1126/science.1133601 CrossRefGoogle Scholar
  10. 10.
    Weiss S (2000) Measuring conformational dynamics of biomolecules by single molecule fluorescence spectroscopy. Nat Struct Mol Biol 7(9):724–729CrossRefGoogle Scholar
  11. 11.
    Greenleaf WJ, Woodside MT, Block SM (2007) High-resolution, single-molecule measurements of biomolecular motion. Annu Rev Biophys Biomol Struct 36:171CrossRefGoogle Scholar
  12. 12.
    Joo C, Balci H, Ishitsuka Y et al (2008) Advances in single-molecule fluorescence methods for molecular biology. Annu Rev Biochem 77:51CrossRefGoogle Scholar
  13. 13.
    Walter NG, Huang CY, Manzo AJ et al (2008) Do-it-yourself guide: how to use the modern single-molecule toolkit. Nat Methods 5:475CrossRefGoogle Scholar
  14. 14.
    Matson SW, Bean DW, George JW (1994) DNA helicases: enzymes with essential roles in all aspects of DNA metabolism. Bioessays 16(1):13–22. doi: 10.1002/bies.950160103 CrossRefGoogle Scholar
  15. 15.
    Schmid SR, Linder P (1992) D-E-A-D protein family of putative RNA helicases. Mol Microbiol 6(3):283–292. doi: 10.1111/j.1365-2958.1992.tb01470.x CrossRefGoogle Scholar
  16. 16.
    Jankowsky E, Gross CH, Shuman S et al (2001) Active disruption of an RNA-protein interaction by a DExH/D RNA helicase. Science 291(5501):121–125. doi: 10.1126/science.291.5501.121 CrossRefGoogle Scholar
  17. 17.
    Lohman TM, Bjornson KP (1996) Mechanisms of helicase-catalyzed DNA unwinding. Annu Rev Biochem 65(1):169–214. doi: 10.1146/ CrossRefGoogle Scholar
  18. 18.
    Tuteja N, Tuteja R (2004) Prokaryotic and eukaryotic DNA helicases. Essential molecular motor proteins for cellular machinery. Eur J Biochem 271(10):1835–1848. doi: 10.1111/j.1432-1033.2004.04093.x CrossRefGoogle Scholar
  19. 19.
    Singleton MR, Dillingham MS, Wigley DB (2007) Structure and mechanism of helicases and nucleic acid translocases. Annu Rev Biochem 76:23–50CrossRefGoogle Scholar
  20. 20.
    Mackintosh SG, Raney KD (2006) DNA unwinding and protein displacement by superfamily 1 and superfamily 2 helicases. Nucleic Acids Res 34(15):4154–4159. doi: 10.1093/nar/gkl501 CrossRefGoogle Scholar
  21. 21.
    Donmez I, Patel SS (2006) Mechanisms of a ring shaped helicase. Nucleic Acids Res 34(15):4216–4224. doi: 10.1093/nar/gkl508 CrossRefGoogle Scholar
  22. 22.
    Jankowsky E, Fairman ME (2007) RNA helicases—one fold for many functions. Curr Opin Struct Biol 17(3):316–324. doi: 10.1016/ CrossRefGoogle Scholar
  23. 23.
    Vindigni A (2007) Biochemical, biophysical, and proteomic approaches to study DNA helicases. Mol Biosyst 3(4):266–274. doi: 10.1039/B616145F Google Scholar
  24. 24.
    Lee KS, Balci H, Jia H et al (2013) Direct imaging of single UvrD helicase dynamics on long single-stranded DNA. Nat Commun 4:1878. doi: 10.1038/ncomms2882 CrossRefGoogle Scholar
  25. 25.
    Lin C-T, Tritschler F, Suk Lee K et al (2014) Single-molecule imaging reveals the translocation dynamics of hepatitis C virus NS3 helicase. Biophys J 106(2):72a. doi: 10.1016/j.bpj.2013.11.474 CrossRefGoogle Scholar
  26. 26.
    Lee KS (2013) Fluorescence imaging of single molecule dynamics on long single stranded DNA. (Doctor of Philosophy Doctoral dissertation), University of Illinois at Urbana-Champaign. Scholar
  27. 27.
    Lee KS, Marciel AB, Kozlov AG et al (2014) Ultrafast redistribution of E. coli SSB along long single-stranded DNA via intersegment transfer. J Mol Biol 426(13):2413–2421. doi: 10.1016/j.jmb.2014.04.023 CrossRefGoogle Scholar
  28. 28.
    Brockman C, Kim SJ, Schroeder CM (2011) Direct observation of single flexible polymers using single stranded DNA. Soft Matter 7(18):8005–8012. doi: 10.1039/C1SM05297G CrossRefGoogle Scholar
  29. 29.
    Zhou R, Schlierf M, Ha T (2010) Chapter Sixteen - Force–fluorescence spectroscopy at the single-molecule level. In: Nils GW (ed) Methods in enzymology, vol 475. Academic, New York, NY, pp 405–426. doi: 10.1016/S0076-6879(10)75016-3 Google Scholar
  30. 30.
    Selvin PR, Ha T (2008) Single-molecule techniques: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  31. 31.
    Hua B, Han KY, Zhou R et al (2014) An improved surface passivation method for single-molecule studies. Nat Methods 11(12):1233–1236. doi: 10.1038/nmeth.3143, - supplementary-informationCrossRefGoogle Scholar
  32. 32.
    Rice SE, Purcell TJ, Spudich JA (2003) [6] Building and using optical traps to study properties of molecular motors. In: Nils GW (ed) Methods in enzymology, vol 361. Academic, New York, NY, pp 112–133. doi: 10.1016/S0076-6879(03)61008-6 Google Scholar
  33. 33.
    Berg-Sørensen K, Flyvbjerg H (2004) Power spectrum analysis for optical tweezers. Rev Sci Instrum 75(3):594–612. doi: 10.1063/1.1645654 CrossRefGoogle Scholar
  34. 34.
    Berg-Sørensen K, Peterman EJG, Weber T et al (2006) Power spectrum analysis for optical tweezers. II: laser wavelength dependence of parasitic filtering, and how to achieve high bandwidth. Rev Sci Instrum 77(6):063106. doi: 10.1063/1.2204589 CrossRefGoogle Scholar
  35. 35.
    Kubo R, Toda M, Hashitsume N (1991) Statistical physics II: nonequilibrium statistical mechanics. Springer, BerlinCrossRefGoogle Scholar
  36. 36.
    Neuman KC, Block SM (2004) Optical trapping. Rev Sci Instrum 75(9):2787–2809. doi: 10.1063/1.1785844 CrossRefGoogle Scholar
  37. 37.
    Toprak E, Balci H, Blehm BH et al (2007) Three-dimensional particle tracking via bifocal imaging. Nano Lett 7(7):2043–2045. doi: 10.1021/nl0709120 CrossRefGoogle Scholar
  38. 38.
    Baumann CG, Smith SB, Bloomfield VA et al (1997) Ionic effects on the elasticity of single DNA molecules. Proc Natl Acad Sci U S A 94(12):6185–6190CrossRefGoogle Scholar
  39. 39.
    Yildiz A, Forkey JN, McKinney SA et al (2003) Myosin V Walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300(5628):2061–2065. doi: 10.1126/science.1084398 CrossRefGoogle Scholar
  40. 40.
    Swoboda M, Henig J, Cheng H-M et al (2012) Enzymatic oxygen scavenging for photostability without pH drop in single-molecule experiments. ACS Nano 6(7):6364–6369. doi: 10.1021/nn301895c CrossRefGoogle Scholar
  41. 41.
    van Dijk MA, Kapitein LC, van Mameren J et al (2004) Combining optical trapping and single-molecule fluorescence spectroscopy: enhanced photobleaching of fluorophores. J Phys Chem B 108(20):6479–6484. doi: 10.1021/jp049805+ CrossRefGoogle Scholar
  42. 42.
    Lang MJ, Asbury CL, Shaevitz JW et al (2002) An automated two-dimensional optical force clamp for single molecule studies. Biophys J 83(1):491–501. doi: 10.1016/S0006-3495(02)75185-0 CrossRefGoogle Scholar
  43. 43.
    Köhler A (1984) New method of illumination for phomicrographical purposes. J R Microsc Soc 14:261–262Google Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Department of Biophysics and Biophysical ChemistryJohns Hopkins UniversityBaltimoreUSA
  2. 2.Department of Biophysics and Biophysical Chemistry, Thomas C. Jenkins Department of Biophysics and Department of Biomedical EngineeringJohns Hopkins UniversityBaltimoreUSA
  3. 3.Howard Hughes Medical InstituteBaltimoreUSA

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