Skip to main content
SpringerLink
Log in

Force-Activated DNA Substrates for In Situ Generation of ssDNA and Designed ssDNA/dsDNA Structures in an Optical-Trapping Assay

  • Protocol
  • First Online:
Optical Tweezers

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2478))

  • 1141 Accesses

Abstract

Single-molecule force spectroscopy can precisely probe the biomechanical interactions of proteins that unwind duplex DNA and bind to and wrap around single-stranded (ss)DNA. Yet assembly of the required substrates, which often contain a ssDNA segment embedded within a larger double-stranded (ds)DNA construct, can be time-consuming and inefficient, particularly when using a standard three-way hybridization protocol. In this chapter, we detail how to construct a variety of force-activated DNA substrates more efficiently. To do so, we engineered a dsDNA molecule with a designed sequence of specified GC content positioned between two enzymatically induced, site-specific nicks. Partially pulling this substrate into the overstretching transition of DNA (~65 pN) using an optical trap led to controlled dissociation of the ssDNA segment delineated by the two nicks. Here, we describe protocols for generating ssDNA of up to 1000 nucleotides as well as more complex structures, such as a 120-base-pair DNA hairpin positioned next to a 33-nucleotide ssDNA segment. The utility of the hairpin substrate was demonstrated by measuring the motion of E. coli. RecQ, a 3′-to-5′ DNA helicase.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Bustamante C, Bryant Z, Smith SB (2003) Ten years of tension: single-molecule DNA mechanics. Nature 421:423–427

    Article  ADS  Google Scholar 

  2. Chemla YR (2010) Revealing the base pair stepping dynamics of nucleic acid motor proteins with optical traps. Phys Chem Chem Phys 12:3080–3095

    Article  Google Scholar 

  3. Chaurasiya KR, Paramanathan T, McCauley MJ, Williams MC (2010) Biophysical characterization of DNA binding from single molecule force measurements. Phys Life Rev 7:299–341

    Article  ADS  Google Scholar 

  4. Zhou J, Schweikhard V, Block SM (2013) Single-molecule studies of RNAPII elongation. Biochim Biophys Acta 1829:29–38

    Article  Google Scholar 

  5. Dumont S, Cheng W, Serebrov V, Beran RK, Tinoco IJ, Pyle AM, Bustamante C (2006) RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP. Nature 439:105–108

    Article  ADS  Google Scholar 

  6. Perkins TT, Li HW, Dalal RV, Gelles J, Block SM (2004) Forward and reverse motion of single RecBCD molecules on DNA. Biophys J 86:1640–1648

    Article  Google Scholar 

  7. Wen JD, Lancaster L, Hodges C, Zeri AC, Yoshimura SH, Noller HF, Bustamante C, Tinoco I (2008) Following translation by single ribosomes one codon at a time. Nature 452:598–603

    Article  ADS  Google Scholar 

  8. Ribeck N, Kaplan DL, Bruck I, Saleh OA (2010) DnaB helicase activity is modulated by DNA geometry and force. Biophys J 99:2170–2179

    Article  Google Scholar 

  9. Manosas M, Xi XG, Bensimon D, Croquette V (2010) Active and passive mechanisms of helicases. Nucleic Acids Res 38:5518–5526

    Article  Google Scholar 

  10. Seol Y, Strub MP, Neuman KC (2016) Single molecule measurements of DNA helicase activity with magnetic tweezers and t-test based step-finding analysis. Methods 105:119–127

    Article  Google Scholar 

  11. Paik DH, Roskens VA, Perkins TT (2013) Torsionally constrained DNA for single-molecule assays: an efficient, ligation-free method. Nucleic Acids Res 41:e179

    Article  Google Scholar 

  12. 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

    Article  Google Scholar 

  13. Perkins TT (2014) Angstrom-precision optical traps and applications. Annu Rev Biophys 43:279–302

    Article  Google Scholar 

  14. Rief M, Clausen-Schaumann H, Gaub HE (1999) Sequence-dependent mechanics of single DNA molecules. Nat Struct Biol 6:346–349

    Article  Google Scholar 

  15. Smith SB, Cui Y, Bustamante C (1996) Overstretching of B-DNA: the elastic response of individual double-stranded and single stranded DNA molecules. Science 271:795–799

    Article  ADS  Google Scholar 

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

    Article  ADS  Google Scholar 

  17. McCauley MJ, Williams MC (2007) Mechanisms of DNA binding determined in optical tweezers experiments. Biopolymers 85:154–168

    Article  Google Scholar 

  18. Paik DH, Seol Y, Halsey WA, Perkins TT (2009) Integrating a high-force optical trap with gold nanoposts and a robust gold-DNA bond. Nano Lett 9:2978–2983

    Article  ADS  Google Scholar 

  19. van Mameren J, Gross P, Farge G, Hooijman P, Modesti M, Falkenberg M, Wuite GJ, Peterman EJ (2009) Unraveling the structure of DNA during overstretching by using multicolor, single-molecule fluorescence imaging. Proc Natl Acad Sci U S A 106:18231–18236

    Article  ADS  Google Scholar 

  20. Zhang X, Chen H, Le S, Rouzina I, Doyle PS, Yan J (2013) Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching by single-molecule calorimetry. Proc Natl Acad Sci U S A 110:3865–3870

    Article  ADS  Google Scholar 

  21. King GA, Gross P, Bockelmann U, Modesti M, Wuite GJ, Peterman EJ (2013) Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching using fluorescence microscopy. Proc Natl Acad Sci U S A 110:3859–3864

    Article  ADS  Google Scholar 

  22. Candelli A, Hoekstra TP, Farge G, Gross P, Peterman EJ, Wuite GJ (2013) A toolbox for generating single-stranded DNA in optical tweezers experiments. Biopolymers 99:611–620

    Article  Google Scholar 

  23. Morfill J, Kuhner F, Blank K, Lugmaier RA, Sedlmair J, Gaub HE (2007) B-S transition in short oligonucleotides. Biophys J 93:2400–2409

    Article  Google Scholar 

  24. Clausen-Schaumann H, Rief M, Tolksdorf C, Gaub HE (2000) Mechanical stability of single DNA molecules. Biophys J 78:1997–2007

    Article  Google Scholar 

  25. Okoniewski SR, Uyetake L, Perkins TT (2017) Force-activated DNA substrates for probing individual proteins interacting with single-stranded DNA. Nucleic Acids Res 45:10775–10782

    Article  Google Scholar 

  26. Walder R, LeBlanc M-A, Van Patten WJ, Edwards DT, Greenbert JA, Adhikari A, Okoniewski SR, Sullan RMA, Rabuka D, Sousa MC, Perkins TT (2017) Rapid characterization of a mechanically labile α-helical protein enabled by efficient site-specific bioconjugation. J Am Chem Soc 139:9867–9875

    Article  Google Scholar 

  27. Sambrook JF, Russell DW (eds) (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor, New York

    Google Scholar 

  28. Okoniewski SR, Carter AR, Perkins TT (2017) A surface-coupled optical trap with 1-bp precision via active stabilization. Methods Mol Biol 1486:77–107

    Article  Google Scholar 

  29. Seol Y, Li J, Nelson PC, Perkins TT, Betterton MD (2007) Elasticity of short DNA molecules: theory and experiment for contour lengths of 0.6-7 μm. Biophys J 93:4360–4373

    Article  Google Scholar 

  30. 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 U S A 103:6190–6195

    Article  ADS  Google Scholar 

  31. Harmon FG, Kowalczykowski SC (1998) RecQ helicase, in concert with RecA and SSB proteins, initiates and disrupts DNA recombination. Genes Dev 12:1134–1144

    Article  Google Scholar 

  32. Eeftens JM, van der Torre J, Burnham DR, Dekker C (2015) Copper-free click chemistry for attachment of biomolecules in magnetic tweezers. BMC Biophys 8:9

    Article  Google Scholar 

  33. Stigler J, Ziegler F, Gieseke A, Gebhardt JCM, Rief M (2011) The complex folding network of single calmodulin molecules. Science 334:512–516

    Article  ADS  Google Scholar 

  34. Kastantin M, Langdon BB, Chang EL, Schwartz DK (2011) Single-molecule resolution of interfacial fibrinogen behavior: effects of oligomer populations and surface chemistry. J Am Chem Soc 133:4975–4983

    Article  Google Scholar 

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

    Article  ADS  Google Scholar 

Download references

Acknowledgments

We thank Keir Neuman and Yeonee Seol for providing RecQ and the DNA constructs to express RecQ. This work was supported by the National Science Foundation (MCB-1716033, Phy-1734006) and the National Institute of Standards and Technology (NIST). Mention of commercial products is for information only; it does not imply NIST’s recommendation or endorsement. T.T.P. is a staff member of NIST’s quantum physics division.

Author information

Authors and Affiliations

  1. JILA, National Institute of Standards and Technology, and University of Colorado, Boulder, CO, USA

    Arnulf M. K. Taylor, Stephen R. Okoniewski, Lyle Uyetake & Thomas T. Perkins

  2. Department of Physics, University of Colorado, Boulder, CO, USA

    Arnulf M. K. Taylor & Stephen R. Okoniewski

  3. Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO, USA

    Thomas T. Perkins

Authors
  1. Arnulf M. K. Taylor
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Stephen R. Okoniewski
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lyle Uyetake
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Thomas T. Perkins
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Thomas T. Perkins .

Editor information

Editors and Affiliations

  1. Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY, USA

    Arne Gennerich

Rights and permissions

Reprints and permissions

Copyright information

© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Taylor, A.M.K., Okoniewski, S.R., Uyetake, L., Perkins, T.T. (2022). Force-Activated DNA Substrates for In Situ Generation of ssDNA and Designed ssDNA/dsDNA Structures in an Optical-Trapping Assay. In: Gennerich, A. (eds) Optical Tweezers. Methods in Molecular Biology, vol 2478. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2229-2_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2229-2_10

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2228-5

  • Online ISBN: 978-1-0716-2229-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation