Force Spectroscopy of DNA and RNA: Structure and Kinetics from Single-Molecule Experiments

  • Rebecca Bolt Ettlinger
  • Michael Askvad Sørensen
  • Lene Broeng OddershedeEmail author
Part of the Nucleic Acids and Molecular Biology book series (NUCLEIC, volume 29)


Force spectroscopy of individual DNA and RNA molecules provides unique insights into the structure and mechanics of these for life so essential molecules. Observations of DNA and RNA molecules one at a time provide spatial, structural, and temporal information that is complementary to the information obtained by classical ensemble methods. Single-molecule force spectroscopy has been realized only within the last decades, and its success is crucially connected to the technological development that has allowed single-molecule resolution. This chapter provides an introduction to in vitro force spectroscopy of individual DNA and RNA molecules including the most commonly used techniques, the theory and methodology necessary for understanding the data, and the exciting results achieved. Three commonly used single-molecule methods are emphasized: optical tweezers, magnetic tweezers, and nanopore force spectroscopy. The theory of DNA stretch and twist under tension is described along with related experimental examples. New principles for extracting kinetic and thermodynamic information from nonequilibrium data are outlined, and further examples are given including the opening of DNA and RNA structures to reveal their energy landscape. Finally, future perspectives for force spectroscopy of DNA and RNA are offered.


Energy Landscape Optical Tweezer Chromatin Fiber Force Spectroscopy Extension Curve 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



Gibbs free energy change


Activation energy


Crooks fluctuation theorem


Double-stranded DNA


Extensible worm-like chain model


Freely jointed chain model


Jarzynski equality


Rate of transition at force F


Rate of transition at zero force




Contour length


Persistence length


Magnetic tweezers


Nanopore force spectroscopy


Optical tweezers


Single-stranded DNA


Twistable worm-like chain model


Worm-like chain model


Distance to the transition state


  1. Alemany A, Mossa A, Junier I, Ritort F (2012) Experimental free-energy measurements of kinetic molecular states using fluctuation theorems. Nat Phys 8:688–694CrossRefGoogle Scholar
  2. Bell GI (1978) Models for the specific adhesion of cells to cells. Science 200(4342):618–627PubMedCrossRefGoogle Scholar
  3. Bizarro CV, Alemany A, Ritort F (2012) Non-specific binding of Na+ and Mg2+ to RNA determined by force spectroscopy methods. Nucleic Acids Res 40(14):6922–6935PubMedCrossRefGoogle Scholar
  4. Boal DH (2002) Mechanics of the cell. Cambridge University Press, CambridgeGoogle Scholar
  5. Bryant Z, Oberstrass FC, Basu A (2012) Recent developments in single-molecule DNA mechanics. Curr Opin Struct Biol 22(3):304–312PubMedCrossRefGoogle Scholar
  6. Bustamante C, Marko JF, Siggia ED, Smith SB (1994) Entropic elasticity of λ-phage DNA. Science 265(5178):1599–1600PubMedCrossRefGoogle Scholar
  7. Bustamante C, Macosko JC, Wuite GJL (2000) Grabbing the cat by the tail: manipulating molecules one by one. Nat Rev Mol Cell Biol 1(2):130–136PubMedCrossRefGoogle Scholar
  8. Chen G, Chang K, Chou M, Bustamante C, Tinoco I Jr (2009) Triplex structures in an RNA pseudoknot enhance mechanical stability and increase efficiency of -1 ribosomal frameshifting. Proc Natl Acad Sci USA 106(31):12706–12711PubMedCrossRefGoogle Scholar
  9. Chen H, Meisburger SP, Pabit SA, Sutton JL, Webb WW, Pollack L (2012) Ionic strength-dependent persistence lengths of single-stranded RNA and DNA. Proc Natl Acad Sci USA 109(3):799–804PubMedCrossRefGoogle Scholar
  10. Cluzel P, Lebrun A, Heller C, Lavery R, Viovy J, Chatenay D, Caron F (1996) DNA: an extensible molecule. Science 271(5250):792–794PubMedCrossRefGoogle Scholar
  11. Collin D, Ritort F, Jarzynski C, Smith SB, Tinoco I Jr, Bustamante C (2005) Verification of the Crooks fluctuation theorem and recovery of RNA folding free energies. Nature 437:231–234PubMedCrossRefGoogle Scholar
  12. Crooks GE (1999) Entropy production fluctuation theorem and the nonequilibrium work relation for free energy differences. Phys Rev E 60(3):2721–2726CrossRefGoogle Scholar
  13. Crut A, Koster DA, Seidel R, Wiggins CH, Dekker NH (2007) Fast dynamics of supercoiled DNA revealed by single-molecule experiments. Proc Natl Acad Sci USA 104(29):11957–11962PubMedCrossRefGoogle Scholar
  14. de Vlaminck I, Dekker C (2012) Recent advances in magnetic tweezers. Annu Rev Biophys 41:453–472PubMedCrossRefGoogle Scholar
  15. Dudko OK, Hummer G, Szabo A (2006) Intrinsic rates and activation free energies from single-molecule pulling experiments. Phys Rev Lett 96:108101PubMedCrossRefGoogle Scholar
  16. Dudko OK, Mathé J, Szabo A, Meller A, Hummer G (2007) Extracting kinetics from single-molecule force spectroscopy: nanopore unzipping of DNA hairpins. Biophys J 92(12):4188–4195PubMedCrossRefGoogle Scholar
  17. Dudko OK, Hummer G, Szabo A (2008) Theory, analysis, and interpretation of single-molecule force spectroscopy experiments. Proc Natl Acad Sci USA 105(41):15755–15760PubMedCrossRefGoogle Scholar
  18. Dudko OK, Mathé J, Meller A (2010) Nanopore force spectroscopy tools for analyzing single biomolecular complexes. Methods Enzym 475:565–589CrossRefGoogle Scholar
  19. Dudko OK, Graham TGW, Best RB (2011) Locating the barrier for folding of single molecules under an external force. Phys Rev Lett 107:208301PubMedCrossRefGoogle Scholar
  20. Essevaz-Roulet B, Bockelmann U, Heslot F (1997) Mechanical separation of the complementary strands of DNA. Proc Natl Acad Sci USA 94(22):11935–11940PubMedCrossRefGoogle Scholar
  21. Gittes F, Schmidt CH (1998) Signals and noise in micromechanical measurements. In: Sheetz MP (ed) Laser tweezers in cell biology, vol 55, Methods in cell biology. Academic, San Diego, pp 129–156Google Scholar
  22. Gore J, Bryant Z, Nöllmann M, Le MU, Cozzarelli NR, Bustamante C (2006) DNA overwinds when stretched. Nature 442(7104):836–839PubMedCrossRefGoogle Scholar
  23. Gross P, Laurens N, Oddershede LB, Bockelmann U, Peterman EJL, Wuite GJL (2011) Quantifying how DNA stretches, melts and changes twist under tension. Nat Phys 7(9):731–736CrossRefGoogle Scholar
  24. Gupta AN, Vincent A, Neupane K, Yu H, Wang F, Woodside MT (2011) Experimental validation of free-energy-landscape reconstruction from non-equilibrium single-molecule force spectroscopy measurements. Nat Phys 7(8):631–634CrossRefGoogle Scholar
  25. Hansen TM, Reihani SNS, Oddershede LB, Sørensen MA (2007) Correlation between mechanical strength of messenger RNA pseudoknots and ribosomal frameshifting. Proc Natl Acad Sci USA 104(14):5830–5835PubMedCrossRefGoogle Scholar
  26. Hummer G, Szabo A (2001) Free energy reconstruction from nonequilibrium single-molecule pulling experiments. Proc Natl Acad Sci USA 98(7):3658–3661PubMedCrossRefGoogle Scholar
  27. Hummer G, Szabo A (2003) Kinetics from nonequilibrium single-molecule pulling experiments. Biophys J 85(1):5–15PubMedCrossRefGoogle Scholar
  28. Hummer G, Szabo A (2010) Free energy profiles from single-molecule pulling experiments. Proc Natl Acad Sci USA 107(50):21441–21446PubMedCrossRefGoogle Scholar
  29. Iwai S, Uyeda TQP (2008) Visualizing myosin-actin interaction with a genetically-encoded fluorescent strain sensor. Proc Natl Acad Sci USA 105(44):16882–16887PubMedCrossRefGoogle Scholar
  30. Jarzynski C (1997) Nonequilibrium equality for free energy differences. Phys Rev Lett 78:2690–2693CrossRefGoogle Scholar
  31. Jin J, Bai L, Johnson DS, Fulbright RM, Kireeva ML, Kashlev M, Wang MD (2010) Synergistic action of RNA polymerases in overcoming the nucleosomal barrier. Nat Struct Mol Biol 17(6):745–752PubMedCrossRefGoogle Scholar
  32. Junier I, Mossa A, Manosas M, Ritort F (2009) Recovery of free energy branches in single molecule experiments. Phys Rev Lett 102:070602PubMedCrossRefGoogle Scholar
  33. Keyser UF, Koeleman BN, Van Dorp S, Krapf D, Smeets RMM, Lemay SD, Dekker NH, Dekker C (2006) Direct force measurements on DNA in a solid-state nanopore. Nat Phys 2(7):473–477CrossRefGoogle Scholar
  34. Killian JL, Sheinia MY, Wang MD (2012) Recent advances in single molecule studies of nucleosomes. Curr Opin Struct Biol 22:80–87PubMedCrossRefGoogle Scholar
  35. Kruithof M, Chien FT, Routh A, Logie C, Rhodes D, van Noort J (2009) Single-molecule force spectroscopy reveals a highly compliant helical folding for the 30-nm chromatin fiber. Nat Struct Mol Biol 16(5):534–540PubMedCrossRefGoogle Scholar
  36. La Porta A, Wang MD (2004) Optical torque wrench: angular trapping, rotation, and torque detection of quartz microparticles. Phys Rev Lett 92:190801PubMedCrossRefGoogle Scholar
  37. Lavelle C, Praly E, Bensimon D, Le Cam E, Croquette V (2011) Nucleosome remodeling machines and other molecular motors observed at the single molecule level. FEBS J 298(19):3596–3607CrossRefGoogle Scholar
  38. Leger JF, Romano G, Sarkar A, Robert J, Bourdieu L, Chatenay D, Marko JF (1999) Structural transitions of a twisted and stretched DNA molecule. Phys Rev Lett 83(5):1066–1069CrossRefGoogle Scholar
  39. Liphardt J, Onoa B, Smith SB, Tinoco I Jr, Bustamante C (2001) Reversible unfolding of single RNA molecules by mechanical force. Science 292(5517):733–737PubMedCrossRefGoogle Scholar
  40. Liphardt J, Dumont S, Smith SB, Tinoco I Jr, Bustamante C (2002) Equilibrium information from nonequilibrium measurements in an experimental test of Jarzynski’s equality. Science 296:1832–1835PubMedCrossRefGoogle Scholar
  41. Mangeol P, Bizebard T, Chiaruttini C, Dreyfus M, Springer M, Bockelmann U (2011) Probing ribosomal protein–RNA interactions with an external force. Proc Natl Acad Sci USA 108(45):18272–18276PubMedCrossRefGoogle Scholar
  42. Marko JF, Siggia ED (1995) Stretching DNA. Macromolecules 28(26):8759–8770CrossRefGoogle Scholar
  43. McNally B, Singer A, Zhiliang Y, Yingjie S, Zhipeng W, Meller A (2010) Optical recognition of converted DNA nucleotides for single-molecule DNA sequencing using nanopore arrays. NanoLetters 10:2237–2244CrossRefGoogle Scholar
  44. Minh DDL, Adib AD (2008) Optimized free energies from bidirectional single-molecule force spectroscopy. Phys Rev Lett 100(18):180602PubMedCrossRefGoogle Scholar
  45. Mossa A, de Lorenzo S, Huguet JM, Ritort F (2009) Measurement of work in single-molecule pulling experiments. J Chem Phys 130:234116PubMedCrossRefGoogle Scholar
  46. Neuman KC, Nagy A (2008) Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy. Nat Methods 5(6):491–505PubMedCrossRefGoogle Scholar
  47. Oddershede LB (2012) Force probing of individual molecules inside the living cell is now a reality. Nat Chem Biol 8:879–886PubMedCrossRefGoogle Scholar
  48. Perkins T, Smith D, Chu S (1997) Single polymer dynamics in an elongational flow. Science 276(5321):2016–2021PubMedCrossRefGoogle Scholar
  49. Ritchie DB, Foster DAN, Woodside MT (2012) Programmed -1 frameshifting efficiency correlates with RNA pseudoknot conformational plasticity, not resistance to mechanical unfolding. Proc Natl Acad Sci USA 109(40):16167–16172PubMedCrossRefGoogle Scholar
  50. Ritort F, Bustamante C, Tinoco I Jr (2002) A two-state kinetic model for the unfolding of single molecules by mechanical force. Proc Natl Acad Sci USA 99(21):13544–13548PubMedCrossRefGoogle Scholar
  51. Rohrbach A (2005) Stiffness of optical traps: quantitative agreement between experiment and electromagnetic theory. Phys Rev Lett 95:168102PubMedCrossRefGoogle Scholar
  52. Rouzina I, Bloomfield VA (2001) Force-induced melting of the DNA double helix 1. Thermodynamic analysis. Biophys J 80(2):882–893PubMedCrossRefGoogle Scholar
  53. Smith SB, Cui Y, Bustamante C (1996) Overstretching B-DNA: the elastic response of individual double stranded and single stranded DNA molecules. Science 271(5250):795–797PubMedCrossRefGoogle Scholar
  54. Stevenson DJ, Gunn-Moore F, Dholakia K (2010) Light forces the pace: optical manipulation for biophotonics. J Biomed Opt 15(2):041503PubMedCrossRefGoogle Scholar
  55. Strick T, Allemand J-F, Bensimon D, Croquette V (1998) The behavior of supercoiled DNA. Biophys J 74:2016–2028PubMedCrossRefGoogle Scholar
  56. Strunz T, Oroszlan K, Schäfer R, Güntherodt HJ (1999) Dynamic force spectroscopy of single DNA molecules. Proc Natl Acad Sci USA 96(20):11277–11282PubMedCrossRefGoogle Scholar
  57. van Mameren J, Gross P, Farge G, Hooijman P, Modesti M, Falkenberg M, Wuite GJL, Peterman EJG (2009) Unraveling the structure of DNA during overstretching by using multicolor, single-molecule fluorescence imaging. Proc Natl Acad Sci USA 106(43):18231–18236PubMedCrossRefGoogle Scholar
  58. Wang MD, Yin H, Landick R, Gelles J, Block SM (1997) Stretching DNA with optical tweezers. Biophys J 72(3):1335–1346PubMedCrossRefGoogle Scholar
  59. Williams MC, Wenner JR, Rouzina I, Bloomfield VA (2001) Effect of ph on the overstretching transition of double-stranded DNA: evidence of force-induced DNA melting. Biophys J 80(2):874–881PubMedCrossRefGoogle Scholar
  60. Woodside MT, Behnke-Parks WM, Larizadeh K, Travers K, Herschlag D, Block SM (2006) Nanomechanical measurements of the sequence-dependent folding landscapes of single nucleic acid hairpins. Proc Natl Acad Sci USA 103(16):6190–6195PubMedCrossRefGoogle Scholar
  61. Zhang X, Chen H, Fu H, Doyle PS, Yan J (2012) Two distinct overstretched DNA structures revealed by single-molecule thermodynamics measurements. Proc Natl Acad Sci USA 109(21):8103–8108PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Rebecca Bolt Ettlinger
    • 1
  • Michael Askvad Sørensen
    • 2
  • Lene Broeng Oddershede
    • 1
    Email author
  1. 1.The Niels Bohr InstituteUniversity of CopenhagenCopenhagenDenmark
  2. 2.The Department of BiologyUniversity of CopenhagenCopenhagenDenmark

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