Abstract
Magnetic tweezers permit application of precisely calibrated stretching forces to nucleic acid molecules tethered between a surface and superparamagnetic beads. In addition, magnetic tweezers can control the tethers’ twist. Here, we focus on recent extensions of the technique that expand the capabilities of conventional magnetic tweezers by enabling direct measurements of single-molecule torque and twist. Magnetic torque tweezers (MTT) still control the DNA or RNA tether’s twist, but directly measure molecular torque by monitoring changes in the equilibrium rotation angle upon overwinding and underwinding of the tether. In freely orbiting magnetic tweezers (FOMT), one end of the tether is allowed to rotate freely, while still applying stretching forces and monitoring rotation angle. Both MTT and FOMT have provided unique insights into the mechanical properties, structural transitions, and interactions of DNA and RNA. Here, we provide step-by-step protocols to carry out FOMT and MTT measurements. In particular, we focus on multiplexed measurements, i.e., measurements that record data for multiple nucleic acid tethers at the same time, to improve statistics and to facilitate the observation of rare events.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Bryant Z, Oberstrass FC, Basu A (2012) Recent developments in single-molecule DNA mechanics. Curr Opin Struct Biol 22:304–312
Lipfert J, van Oene MM, Lee M, Pedaci F, Dekker NH (2015) Torque spectroscopy for the study of rotary motion in biological systems. Chem Rev 115:1449–1474
Neuman KC, Nagy A (2008) Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy. Nat Methods 5:491–505
Manosas M, Meglio A, Spiering MM, Ding F, Benkovic SJ, Barre F-X et al (2010) Magnetic tweezers for the study of DNA tracking motors. Methods Enzymol 475:297–320
Strick TR, Allemand JF, Bensimon D, Bensimon A, Croquette V (1996) The elasticity of a single supercoiled DNA molecule. Science 271:1835–1837
Gosse C, Croquette V (2002) Magnetic tweezers: micromanipulation and force measurement at the molecular level. Biophys J 82:3314–3329
te Velthuis AJW, Kerssemakers JWJ, Lipfert J, Dekker NH (2010) Quantitative guidelines for force calibration through spectral analysis of magnetic tweezers data. Biophys J 99:1292–1302
Lansdorp BM, Saleh OA (2012) Power spectrum and Allan variance methods for calibrating single-molecule video-tracking instruments. Rev Sci Instrum 83:025115
Daldrop P, Brutzer H, Huhle A, Kauert DJ, Seidel R (2015) Extending the range for force calibration in magnetic tweezers. Biophys J 108:2550–2561
Strick TR, Allemand JF, Bensimon D, Croquette V (2000) Stress-induced structural transitions in DNA and proteins. Annu Rev Biophys Biomol Struct 29:523–543
Abels JA, Moreno-Herrero F, Van Der Heijden T, Dekker C, Dekker NH (2005) Single-molecule measurements of the persistence length of double-stranded RNA. Biophys J 88:2737–2744
Herrero-Galán E, Fuentes-Perez ME, Carrasco C, Valpuesta JM, Carrascosa JL, Moreno-Herrero F et al (2013) Mechanical identities of RNA and DNA double helices unveiled at the single-molecule level. J Am Chem Soc 135:122–131
Lipfert J, Klijnhout S, Dekker NH (2010) Torsional sensing of small-molecule binding using magnetic tweezers. Nucleic Acids Res 38:7122–7132
Salerno D, Brogioli D, Cassina V, Turchi D, Beretta GL, Seruggia D et al (2010) Magnetic tweezers measurements of the nanomechanical properties of DNA in the presence of drugs. Nucleic Acids Res 38:7089–7099
Wang Y, Schellenberg H, Walhorn V, Toensing K, Anselmetti D (2017) Binding mechanism of fluorescent dyes to DNA characterized by magnetic tweezers. Mater Today Proc 4:S218–S225
Vilfan ID, Lipfert J, Koster DA, Lemay SG, Dekker NH (2009) Magnetic tweezers for single molecule measurements. Springer, New York, NY
Carrasco C, Dillingham MS, Moreno-Herrero F (2014) Single molecule approaches to monitor the recognition and resection of double-stranded DNA breaks during homologous recombination. DNA Repair (Amst) 20:119–129
Dulin D, Berghuis BA, Depken M, Dekker NH (2015) Untangling reaction pathways through modern approaches to high-throughput single-molecule force-spectroscopy experiments. Curr Opin Struct Biol 34:116–122
Berghuis BA, Köber M, van Laar T, Dekker NH (2016) High-throughput, high-force probing of DNA-protein interactions with magnetic tweezers. Methods 105:90–98
Ordu O, Lusser A, Dekker NH (2016) Recent insights from in vitro single-molecule studies into nucleosome structure and dynamics. Biophys Rev 8:33–49
Hodeib S, Raj S, Manosas M, Zhang W, Bagchi D, Ducos B et al (2016) Single molecule studies of helicases with magnetic tweezers. Methods 105:3–15
Bryant Z, Stone MD, Gore J, Smith SB, Cozzarelli NR, Bustamante C (2003) Structural transitions and elasticity from torque measurements on DNA. Nature 424:338–341
Gore J, Bryant Z, Stone MD, Nöllmann M, Cozzarelli NR, Bustamante C (2006) Mechanochemical analysis of DNA gyrase using rotor bead tracking. Nature 439:100–104
Lebel P, Basu A, Oberstrass FC, Tretter EM, Bryant Z (2014) Gold rotor bead tracking for high-speed measurements of DNA twist, torque and extension. Nat Methods 11:456–462
Oberstrass FC, Fernandes LE, Bryant Z (2012) Torque measurements reveal sequence-specific cooperative transitions in supercoiled DNA. Proc Natl Acad Sci U S A 109:6106–6111
Vlijm R, Lee M, Lipfert J, Lusser A, Dekker C, Dekker NH (2015) Nucleosome assembly dynamics involve spontaneous fluctuations in the handedness of Tetrasomes. Cell Rep 10:216–225
Lipfert J, Wiggin M, Kerssemakers JWJ, Pedaci F, Dekker NH (2011) Freely orbiting magnetic tweezers to directly monitor changes in the twist of nucleic acids. Nat Commun 2:439–439
Lee M, Lipfert J, Sanchez H, Wyman C, Dekker NH (2013) Structural and torsional properties of the RAD51-dsDNA nucleoprotein filament. Nucleic Acids Res 41:7023–7030
Harada Y, Ohara O, Takatsuki A, Itoh H, Shimamoto N, Kinosita K (2001) Direct observation of DNA rotation during transcription by Escherichia coli RNA polymerase. Nature 409:113–115
Basu A, Schoeffler AJ, Berger JM, Bryant Z (2012) ATP binding controls distinct structural transitions of Escherichia coli DNA gyrase in complex with DNA. Nat Struct Mol Biol 19:538–546
Celedon A, Nodelman IM, Wildt B, Dewan R, Searson P, Wirtz D et al (2009) Magnetic tweezers measurement of single molecule torque. Nano Lett 9:1720–1725
Celedon A, Wirtz D, Sun S (2010) Torsional mechanics of DNA are regulated by small-molecule intercalation. J Phys Chem B 114:16929–16935
Lipfert J, Kerssemakers JWJ, Jager T, Dekker NH (2010) Magnetic torque tweezers: measuring torsional stiffness in DNA and RecA-DNA filaments. Nat Methods 7:977–980
Lipfert J, Kerssemakers JJW, Rojer M, Dekker NH (2011) A method to track rotational motion for use in single-molecule biophysics. Rev Sci Instrum 82:103707
Janssen XJA, Lipfert J, Jager T, Daudey R, Beekman J, Dekker NH (2012) Electromagnetic torque tweezers: a versatile approach for measurement of single-molecule twist and torque. Nano Lett 12:3634–3639
Mosconi F, Allemand JF, Croquette V (2011) Soft magnetic tweezers: a proof of principle. Rev Sci Instrum 82:034302
Kauert DJ, Kurth T, Liedl T, Seidel R (2011) Direct mechanical measurements reveal the material properties of three-dimensional DNA origami. Nano Lett 11:5558–5563
Lipfert J, Skinner GM, Keegstra JM, Hensgens T, Jager T, Dulin D et al (2014) Double-stranded RNA under force and torque: similarities to and striking differences from double-stranded DNA. Proc Natl Acad Sci U S A 111:15408–15413
Lipfert J, Lee M, Ordu O, Kerssemakers JWJ, Dekker NH (2014) Magnetic tweezers for the measurement of twist and torque. J Vis Exp 19(87). https://doi.org/10.3791/51503
van Oene MM, Dickinson LE, Pedaci F, Köber M, Dulin D, Lipfert J et al (2015) Biological magnetometry: torque on superparamagnetic beads in magnetic fields. Phys Rev Lett 114:218301. https://doi.org/10.1103/PhysRevLett.114.218301
van Oene MM, Dickinson LE, Cross B, Pedaci F, Lipfert J, Dekker NH (2017) Applying torque to the Escherichia coli flagellar motor using magnetic tweezers. Sci Rep 7:43285
Nord AL, Gachon E, Perez-Carrasco R, Nirody JA, Barducci A, Berry RM et al (2017) Catch bond drives stator mechanosensitivity in the bacterial flagellar motor. Proc Natl Acad Sci U S A 114:12952–12957
Ribeck N, Saleh OA (2008) Multiplexed single-molecule measurements with magnetic tweezers. Rev Sci Instrum 79:094301
De Vlaminck I, Henighan T, van Loenhout MTJ, Pfeiffer I, Huijts J, Kerssemakers JWJ et al (2011) Highly parallel magnetic tweezers by targeted DNA tethering. Nano Lett 11:5489–5493
Cnossen JP, Dulin D, Dekker NH (2014) An optimized software framework for real-time, high-throughput tracking of spherical beads. Rev Sci Instrum 85:103712
Kriegel F, Ermann N, Forbes R, Dulin D, Dekker NH, Lipfert J (2017) Probing the salt dependence of the torsional stiffness of DNA by multiplexed magnetic torque tweezers. Nucleic Acids Res 45:5920–5929
Nomidis SK, Kriegel F, Vanderlinden W, Lipfert J, Carlon E (2017) Twist-bend coupling and the torsional response of double-stranded DNA. Phys Rev Lett 118:217801
Dulin D, Vilfan ID, Berghuis BA, Hage S, Bamford DH, Poranen MM et al (2015) Elongation-competent pauses govern the Fidelity of a viral RNA-dependent RNA polymerase. Cell Rep 10:983–992
van Loenhout MTJ, Kerssemakers JWJ, De Vlaminck I, Dekker C (2012) Non-bias-limited tracking of spherical particles, enabling Nanometer resolution at low magnification. Biophys J 102:2362–2371
Lipfert J, Koster DA, Vilfan ID, Hage S, Dekker NH (2009) Single-molecule magnetic tweezers studies of type IB topoisomerases. Methods Mol Biol 582:71–89
Lipfert J, Hao X, Dekker NH (2009) Quantitative modeling and optimization of magnetic tweezers. Biophys J 96:5040–5049
De Vlaminck I, Henighan T, van Loenhout MT, Burnham DR, Dekker C (2012) Magnetic forces and DNA mechanics in multiplexed magnetic tweezers. PLoS One 7:e41432
Klaue D, Seidel R (2009) Torsional stiffness of single Superparamagnetic microspheres in an external magnetic field. Phys Rev Lett 102:028302
Kriegel F, Ermann N, Lipfert J (2017) Probing the mechanical properties, conformational changes, and interactions of nucleic acids with magnetic tweezers. J Struct Biol 197:26–36
Mosconi F, Allemand JF, Bensimon D, Croquette V (2009) Measurement of the torque on a single stretched and twisted DNA using magnetic tweezers. Phys Rev Lett 102:078301
Sheinin MY, Forth S, Marko JF, Wang MD (2011) Underwound DNA under tension: structure, elasticity, and sequence-dependent Behaviors. Phys Rev Lett 107:108102
Acknowledgments
We thank Jelle van der Does for help with instrument development, Susanne Hage for help with development of the DNA construct, Philipp Walker for instrument construction and useful discussions, the Rief chair at the TU Munich for use of the laser cutter, and the German Research Foundation (DFG) via Sonderforschungbereich SFB 863 “Forces in Biomolecular Systems” for funding.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Kriegel, F., Vanderlinden, W., Nicolaus, T., Kardinal, A., Lipfert, J. (2018). Measuring Single-Molecule Twist and Torque in Multiplexed Magnetic Tweezers. In: Lyubchenko, Y. (eds) Nanoscale Imaging. Methods in Molecular Biology, vol 1814. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8591-3_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8591-3_6
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8590-6
Online ISBN: 978-1-4939-8591-3
eBook Packages: Springer Protocols