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Nucleic Acid Nanotechnology pp 23–52Cite as

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

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Part of the book series: Nucleic Acids and Molecular Biology ((NUCLEIC,volume 29))

Abstract

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.

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Abbreviations

ΔG :

Gibbs free energy change

ΔG ‡ :

Activation energy

CFT:

Crooks fluctuation theorem

dsDNA:

Double-stranded DNA

EWLC:

Extensible worm-like chain model

FJC:

Freely jointed chain model

JE:

Jarzynski equality

k(F):

Rate of transition at force F

k 0 :

Rate of transition at zero force

K 0 :

Elasticity

L c :

Contour length

L p :

Persistence length

MT:

Magnetic tweezers

NFS:

Nanopore force spectroscopy

OT:

Optical tweezers

ssDNA:

Single-stranded DNA

TWLC:

Twistable worm-like chain model

WLC:

Worm-like chain model

x ‡ :

Distance to the transition state

References

  • Alemany A, Mossa A, Junier I, Ritort F (2012) Experimental free-energy measurements of kinetic molecular states using fluctuation theorems. Nat Phys 8:688–694

    Article  CAS  Google Scholar 

  • Bell GI (1978) Models for the specific adhesion of cells to cells. Science 200(4342):618–627

    Article  PubMed  CAS  Google Scholar 

  • 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):6922–6935

    Article  PubMed  CAS  Google Scholar 

  • Boal DH (2002) Mechanics of the cell. Cambridge University Press, Cambridge

    Google Scholar 

  • Bryant Z, Oberstrass FC, Basu A (2012) Recent developments in single-molecule DNA mechanics. Curr Opin Struct Biol 22(3):304–312

    Article  PubMed  CAS  Google Scholar 

  • Bustamante C, Marko JF, Siggia ED, Smith SB (1994) Entropic elasticity of λ-phage DNA. Science 265(5178):1599–1600

    Article  PubMed  CAS  Google Scholar 

  • 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):130–136

    Article  PubMed  CAS  Google Scholar 

  • 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):12706–12711

    Article  PubMed  CAS  Google Scholar 

  • 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):799–804

    Article  PubMed  CAS  Google Scholar 

  • Cluzel P, Lebrun A, Heller C, Lavery R, Viovy J, Chatenay D, Caron F (1996) DNA: an extensible molecule. Science 271(5250):792–794

    Article  PubMed  CAS  Google Scholar 

  • 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 437:231–234

    Article  PubMed  CAS  Google Scholar 

  • Crooks GE (1999) Entropy production fluctuation theorem and the nonequilibrium work relation for free energy differences. Phys Rev E 60(3):2721–2726

    Article  CAS  Google Scholar 

  • 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):11957–11962

    Article  PubMed  CAS  Google Scholar 

  • de Vlaminck I, Dekker C (2012) Recent advances in magnetic tweezers. Annu Rev Biophys 41:453–472

    Article  PubMed  Google Scholar 

  • Dudko OK, Hummer G, Szabo A (2006) Intrinsic rates and activation free energies from single-molecule pulling experiments. Phys Rev Lett 96:108101

    Article  PubMed  Google Scholar 

  • 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):4188–4195

    Article  PubMed  CAS  Google Scholar 

  • Dudko OK, Hummer G, Szabo A (2008) Theory, analysis, and interpretation of single-molecule force spectroscopy experiments. Proc Natl Acad Sci USA 105(41):15755–15760

    Article  PubMed  CAS  Google Scholar 

  • Dudko OK, Mathé J, Meller A (2010) Nanopore force spectroscopy tools for analyzing single biomolecular complexes. Methods Enzym 475:565–589

    Article  CAS  Google Scholar 

  • Dudko OK, Graham TGW, Best RB (2011) Locating the barrier for folding of single molecules under an external force. Phys Rev Lett 107:208301

    Article  PubMed  Google Scholar 

  • Essevaz-Roulet B, Bockelmann U, Heslot F (1997) Mechanical separation of the complementary strands of DNA. Proc Natl Acad Sci USA 94(22):11935–11940

    Article  PubMed  CAS  Google Scholar 

  • 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 129–156

    Google Scholar 

  • Gore J, Bryant Z, Nöllmann M, Le MU, Cozzarelli NR, Bustamante C (2006) DNA overwinds when stretched. Nature 442(7104):836–839

    Article  PubMed  CAS  Google Scholar 

  • 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):731–736

    Article  CAS  Google Scholar 

  • 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):631–634

    Article  CAS  Google Scholar 

  • 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):5830–5835

    Article  PubMed  CAS  Google Scholar 

  • Hummer G, Szabo A (2001) Free energy reconstruction from nonequilibrium single-molecule pulling experiments. Proc Natl Acad Sci USA 98(7):3658–3661

    Article  PubMed  CAS  Google Scholar 

  • Hummer G, Szabo A (2003) Kinetics from nonequilibrium single-molecule pulling experiments. Biophys J 85(1):5–15

    Article  PubMed  CAS  Google Scholar 

  • Hummer G, Szabo A (2010) Free energy profiles from single-molecule pulling experiments. Proc Natl Acad Sci USA 107(50):21441–21446

    Article  PubMed  CAS  Google Scholar 

  • Iwai S, Uyeda TQP (2008) Visualizing myosin-actin interaction with a genetically-encoded fluorescent strain sensor. Proc Natl Acad Sci USA 105(44):16882–16887

    Article  PubMed  CAS  Google Scholar 

  • Jarzynski C (1997) Nonequilibrium equality for free energy differences. Phys Rev Lett 78:2690–2693

    Article  CAS  Google Scholar 

  • 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):745–752

    Article  PubMed  CAS  Google Scholar 

  • Junier I, Mossa A, Manosas M, Ritort F (2009) Recovery of free energy branches in single molecule experiments. Phys Rev Lett 102:070602

    Article  PubMed  Google Scholar 

  • 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):473–477

    Article  CAS  Google Scholar 

  • Killian JL, Sheinia MY, Wang MD (2012) Recent advances in single molecule studies of nucleosomes. Curr Opin Struct Biol 22:80–87

    Article  PubMed  CAS  Google Scholar 

  • 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):534–540

    Article  PubMed  CAS  Google Scholar 

  • La Porta A, Wang MD (2004) Optical torque wrench: angular trapping, rotation, and torque detection of quartz microparticles. Phys Rev Lett 92:190801

    Article  PubMed  Google Scholar 

  • 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):3596–3607

    Article  Google Scholar 

  • 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):1066–1069

    Article  CAS  Google Scholar 

  • Liphardt J, Onoa B, Smith SB, Tinoco I Jr, Bustamante C (2001) Reversible unfolding of single RNA molecules by mechanical force. Science 292(5517):733–737

    Article  PubMed  CAS  Google Scholar 

  • 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 296:1832–1835

    Article  PubMed  CAS  Google Scholar 

  • 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):18272–18276

    Article  PubMed  CAS  Google Scholar 

  • Marko JF, Siggia ED (1995) Stretching DNA. Macromolecules 28(26):8759–8770

    Article  CAS  Google Scholar 

  • 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 10:2237–2244

    Article  CAS  Google Scholar 

  • Minh DDL, Adib AD (2008) Optimized free energies from bidirectional single-molecule force spectroscopy. Phys Rev Lett 100(18):180602

    Article  PubMed  Google Scholar 

  • Mossa A, de Lorenzo S, Huguet JM, Ritort F (2009) Measurement of work in single-molecule pulling experiments. J Chem Phys 130:234116

    Article  PubMed  Google Scholar 

  • Neuman KC, Nagy A (2008) Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy. Nat Methods 5(6):491–505

    Article  PubMed  CAS  Google Scholar 

  • Oddershede LB (2012) Force probing of individual molecules inside the living cell is now a reality. Nat Chem Biol 8:879–886

    Article  PubMed  CAS  Google Scholar 

  • Perkins T, Smith D, Chu S (1997) Single polymer dynamics in an elongational flow. Science 276(5321):2016–2021

    Article  PubMed  CAS  Google Scholar 

  • 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):16167–16172

    Article  PubMed  CAS  Google Scholar 

  • 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):13544–13548

    Article  PubMed  CAS  Google Scholar 

  • Rohrbach A (2005) Stiffness of optical traps: quantitative agreement between experiment and electromagnetic theory. Phys Rev Lett 95:168102

    Article  PubMed  Google Scholar 

  • Rouzina I, Bloomfield VA (2001) Force-induced melting of the DNA double helix 1. Thermodynamic analysis. Biophys J 80(2):882–893

    Article  PubMed  CAS  Google Scholar 

  • 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):795–797

    Article  PubMed  CAS  Google Scholar 

  • Stevenson DJ, Gunn-Moore F, Dholakia K (2010) Light forces the pace: optical manipulation for biophotonics. J Biomed Opt 15(2):041503

    Article  PubMed  Google Scholar 

  • Strick T, Allemand J-F, Bensimon D, Croquette V (1998) The behavior of supercoiled DNA. Biophys J 74:2016–2028

    Article  PubMed  CAS  Google Scholar 

  • Strunz T, Oroszlan K, Schäfer R, Güntherodt HJ (1999) Dynamic force spectroscopy of single DNA molecules. Proc Natl Acad Sci USA 96(20):11277–11282

    Article  PubMed  CAS  Google Scholar 

  • 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):18231–18236

    Article  PubMed  Google Scholar 

  • Wang MD, Yin H, Landick R, Gelles J, Block SM (1997) Stretching DNA with optical tweezers. Biophys J 72(3):1335–1346

    Article  PubMed  CAS  Google Scholar 

  • 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):874–881

    Article  PubMed  CAS  Google Scholar 

  • 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):6190–6195

    Article  PubMed  CAS  Google Scholar 

  • 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):8103–8108

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. The Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark

    Rebecca Bolt Ettlinger & Lene Broeng Oddershede

  2. The Department of Biology, University of Copenhagen, Copenhagen, Denmark

    Michael Askvad Sørensen

Authors
  1. Rebecca Bolt Ettlinger
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  2. Michael Askvad Sørensen
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  3. Lene Broeng Oddershede
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Corresponding author

Correspondence to Lene Broeng Oddershede .

Editor information

Editors and Affiliations

  1. Dept. of Molecular Biology, University of Aarhus, Center for DNA Nanotechnology, Interdisciplinary Nanoscience Center, Aarhus C, Denmark

    Jørgen Kjems

  2. Center for DNA Nanotechnology, Interdisciplinary Nanoscience Center, Aarhus University, Aarhus C, Denmark

    Elena Ferapontova

  3. Center for DNA Nanotechnology, Interdisciplinary Nanoscience Center and Department of Chemistry, Aarhus University, Aarhus C, Denmark

    Kurt V. Gothelf

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Ettlinger, R.B., Sørensen, M.A., Oddershede, L.B. (2014). Force Spectroscopy of DNA and RNA: Structure and Kinetics from Single-Molecule Experiments. In: Kjems, J., Ferapontova, E., Gothelf, K. (eds) Nucleic Acid Nanotechnology. Nucleic Acids and Molecular Biology, vol 29. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-38815-6_2

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