When double-stranded DNA is stretched to roughly 65 pN it elongates about 1.7-fold, and in torsion-unconstrained molecules this overstretching is partitioned among three competing structures: S-DNA (an internally base-paired elongated duplex), peeled single-stranded DNA unzipped from nicks or free ends, and internal melting bubbles. Zhang et al. (JACS 2014) showed that these structures interconvert when NaCl concentration is changed at constant force, and that reannealing from peeled ssDNA to S-DNA is ultraslow, which they attributed to secondary hairpins forming on the relaxed peeled strand. The unresolved problem is a quantitative, microscopic characterization of this interconversion network: (1) whether and how S-DNA base-pair stability and B-to-S kinetics vary along heterogeneous DNA sequences, including whether apparently smooth B-to-S transitions hide fast sequence-dependent steps; (2) the microscopic mechanism and free-energy landscape of B-to-S and S-to-B conversion; (3) the kinetics and thermodynamics of intramolecular hairpin formation on the peeled strand as a kinetic trap; and (4) the kinetics and pathways of reannealing from peeled ssDNA at very high salt (around 1000 mM NaCl). Later work — molecular-dynamics free-energy profiles, AT-tract-controlled reannealing steps, base-analog studies of short oligomers, and direct visualization with hybrid instruments combining high-speed atomic force microscopy (AFM) and optical tweezers — has advanced restricted pieces of this network, but a 2025 study states that systematic analysis of the sequence dependence of the B-to-S transition is still lacking, and the hairpin trap and high-salt reannealing pathways remain unquantified.

Background

In single-molecule force spectroscopy, a DNA molecule is tethered between a surface and a force-measuring probe (optical tweezers trap a micron-sized bead in a laser focus; magnetic tweezers pull on a paramagnetic bead), so that extension can be monitored while a calibrated tension is applied. Smith, Cui and Bustamante (Science, 1996) discovered that double-stranded B-form DNA, the canonical right-handed helix, undergoes a cooperative overstretching transition near 65 pN in which its contour length increases by about 70%. For torsion-unconstrained molecules — DNA whose strands are free to rotate, because of nicks (single-strand breaks in the sugar-phosphate backbone) or freely swiveling attachment points — later work showed that this plateau is not a single structure. Fu, Chen, Marko and Yan (Nucleic Acids Research, 2010) identified two distinct overstretched states, and Paik and Perkins (JACS, 2011) showed overstretching can occur without peeling from free ends or nicks. Bosaeus et al. (PNAS, 2012) demonstrated with GC-rich short oligomers that a base-paired overstretched duplex, called S-DNA, exists. Two 2013 PNAS studies — Zhang, Chen, Le, Rouzina, Doyle and Yan (single-molecule calorimetry, i.e. inferring the heat and entropy changes of the transition from simultaneous force and extension measurements) and King, Gross, Bockelmann, Modesti, Wuite and Peterman (fluorescence microscopy) — established that overstretching is a competition among three structures: S-DNA, peeled ssDNA (one strand peeled away starting from a nick or free end, leaving the other strand relaxed), and internal melting bubbles (locally separated base-paired regions with intact strands elsewhere).

Zhang, Qu, Chen, Rouzina, Zhang, Doyle and Yan (JACS 136, 16073, 2014) then studied interconversion among these overstretched structures directly: holding DNA at constant forces above 70 pN while stepping the NaCl concentration, they observed stepwise interconversions between S-DNA and melting bubbles in end-closed constructs, and between S-DNA and peeled ssDNA in end-opened constructs (in end-closed constructs the complementary strand ends are covalently joined so that peeling cannot nucleate, while end-opened constructs leave free ends or nicks through which a strand can peel). Two findings define the open problem. First, the B-to-S transition itself appeared smooth, with no detectable stepwise dynamics at their temporal resolution, so any sequence dependence of the transition kinetics along the heterogeneous sequence must either be weak or occur faster than the measurement bandwidth. Second, conversion from peeled ssDNA to S-DNA has ultraslow kinetics, which the authors explained phenomenologically by formation of intramolecular secondary hairpins on the relaxed peeled strand — a kinetic trap — supported by the salt and topology dependence, but without direct visualization or quantitative modeling. Reannealing from peeled ssDNA at very high salt (around 1000 mM NaCl) was slower than the sampled observation windows, so its pathway was left undetermined. Throughout, 'S-DNA' denotes the elongated, still base-paired duplex under tension (sometimes called Σ-DNA), distinct from force-melted single strands; AT and GC content denote the fraction of adenine–thymine versus guanine–cytosine base pairs, which controls base-pair stability.

Problem Statement

Determine, at microscopic (base-pair) resolution, the mechanisms, kinetics, and thermodynamics that govern interconversion among the overstretched structures of torsion-unconstrained double-stranded DNA — S-DNA, peeled single-stranded DNA, and internal melting bubbles — following the interconversion studies of Zhang et al. (2014). This open problem decomposes into four linked questions about this one interconversion network, each grounded in that study's observations and each still unresolved in the later literature cited in ## References:

  1. Sequence-dependent S-DNA stability and B-to-S kinetics: establish whether and how the base-pair stability and energetics of S-DNA vary along heterogeneous DNA sequences, and whether sequence heterogeneity modulates B-to-S transition kinetics. In particular, determine whether the apparent smoothness of B-to-S transitions at conventional single-molecule temporal resolution reflects genuinely weak sequence sensitivity or rapid, small-scale sequence-dependent steps below that resolution.

  2. Microscopic mechanism and free-energy landscape: determine the microscopic pathway and the free-energy landscape of the B-to-S and S-to-B conversions, including the nature of the transition state and the cooperative unit, and their dependence on sequence, salt concentration, and topology.

  3. Hairpin kinetic trap: quantitatively characterize the kinetics and thermodynamics of intramolecular secondary-structure (hairpin) formation on the relaxed peeled strand, and determine whether (and to what extent) it acts as the kinetic trap for the ultraslow reannealing of peeled ssDNA to S-DNA (and to B-DNA at very high salt).

  4. High-salt reannealing: determine the kinetics and the molecular pathways by which reannealing from peeled ssDNA completes at very high salt (on the order of 1000 mM NaCl), including whether alternative intermediates occur on the way back to B-DNA or S-DNA.

All four questions are mandatory parts of the one interconversion network, and together they exhaust the task: the S-DNA-to-melting-bubble interconversion branch is context for that network, not an additional required deliverable. Resolving any one of the four alone is a partial result. For each, a resolution requires a quantitative mechanism, rate, or free-energy profile rather than a proxy observable. Answers may be experimental, theoretical, or simulation-based, or combine them, but must resolve these physical questions themselves rather than only proxy observables, and must retain generality over heterogeneous DNA sequences and long DNA molecules.

References

  1. Xinghua Zhang, Yuanyuan Qu, Hu Chen, Ioulia Rouzina, Shengli Zhang, Patrick S. Doyle, Jie Yan (2014). Interconversion between Three Overstretched DNA Structures. Journal of the American Chemical Society 136, 16073–16080. DOI 10.1021/ja5090805, https://doi.org/10.1021/ja5090805 (source paper)
  2. Steven B. Smith, Yujia Cui, Carlos Bustamante (1996). Overstretching B-DNA: The Elastic Response of Individual Double-Stranded and Single-Stranded DNA Molecules. Science 271, 795–799. DOI 10.1126/science.271.5250.795, https://doi.org/10.1126/science.271.5250.795
  3. Hongxia Fu, Hu Chen, John F. Marko, Jie Yan (2010). Two distinct overstretched DNA states. Nucleic Acids Research 38, 5594–5600. DOI 10.1093/nar/gkq309, https://doi.org/10.1093/nar/gkq309
  4. D. Hern Paik, Thomas T. Perkins (2011). Overstretching DNA at 65 pN Does Not Require Peeling from Free Ends or Nicks. Journal of the American Chemical Society 133, 3219–3221. DOI 10.1021/ja108952v, https://doi.org/10.1021/ja108952v
  5. Niklas Bosaeus, Afaf H. El-Sagheer, Tom Brown, Steven B. Smith, Björn Åkerman, Carlos Bustamante, Bengt Nordén (2012). Tension induces a base-paired overstretched DNA conformation. Proceedings of the National Academy of Sciences 109, 15179–15184. DOI 10.1073/pnas.1213172109, https://doi.org/10.1073/pnas.1213172109
  6. Xinghua Zhang, Hu Chen, Shimin Le, Ioulia Rouzina, Patrick S. Doyle, Jie Yan (2013). Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching by single-molecule calorimetry. Proceedings of the National Academy of Sciences 110, 3865–3870. DOI 10.1073/pnas.1213740110, https://doi.org/10.1073/pnas.1213740110
  7. Graeme A. King, Peter Gross, Ulrich Bockelmann, Mauro Modesti, Gijs J. L. Wuite, Erwin J. G. Peterman (2013). Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching using fluorescence microscopy. Proceedings of the National Academy of Sciences 110, 3859–3864. DOI 10.1073/pnas.1213676110, https://doi.org/10.1073/pnas.1213676110
  8. Lorenzo Bongini, Luca Melli, Vincenzo Lombardi, Pasquale Bianco (2014). Transient kinetics measured with force steps discriminate between double-stranded DNA elongation and melting and define the reaction energetics. Nucleic Acids Research 42, 3436–3449. DOI 10.1093/nar/gkt1297, https://doi.org/10.1093/nar/gkt1297
  9. Lorenzo Bongini, Vincenzo Lombardi, Pasquale Bianco (2014). The transition mechanism of DNA overstretching: a microscopic view using molecular dynamics. Journal of the Royal Society Interface 11, 20140399. DOI 10.1098/rsif.2014.0399, https://doi.org/10.1098/rsif.2014.0399
  10. Niklas Bosaeus, Afaf H. El-Sagheer, Tom Brown, Björn Åkerman, Bengt Nordén (2014). Force-induced melting of DNA—evidence for peeling and internal melting from force spectra on short synthetic duplex sequences. Nucleic Acids Research 42, 8083–8091. DOI 10.1093/nar/gku441, https://doi.org/10.1093/nar/gku441
  11. You-Liang Zhu, Zhong-Yuan Lu, Zhao-Yan Sun (2016). The mechanism of the emergence of distinct overstretched DNA states. The Journal of Chemical Physics 144, 024901. DOI 10.1063/1.4939623, https://doi.org/10.1063/1.4939623
  12. ZhouJie Yang, GuoHua Yuan, WeiLi Zhai, Jie Yan, Hu Chen (2016). The kinetics of force-dependent hybridization and strand-peeling of short DNA fragments. Science China Physics, Mechanics & Astronomy 59, 680013. DOI 10.1007/s11433-016-0112-0, https://doi.org/10.1007/s11433-016-0112-0
  13. Lorenzo Bongini, C. Pongor, G. Falorsi, I. Pertici, M. Kellermayer, Vincenzo Lombardi, Pasquale Bianco (2016). An AT-barrier mechanically controls DNA reannealing under tension. Nucleic Acids Research 44, 7954–7962. DOI 10.1093/nar/gkw604, https://doi.org/10.1093/nar/gkw604
  14. Ashok Garai, Santosh Mogurampelly, Saientan Bag, Prabal K. Maiti (2017). Overstretching of B-DNA with various pulling protocols: Appearance of structural polymorphism and S-DNA. The Journal of Chemical Physics 147, 225102. DOI 10.1063/1.4991862, arXiv:1711.08880, https://doi.org/10.1063/1.4991862
  15. Niklas Bosaeus, Anna Reymer, Tamás Beke-Somfai, Tom Brown, Masayuki Takahashi, Pernilla Wittung-Stafshede, Sandra Rocha, Bengt Nordén (2017). A stretched conformation of DNA with a biological role? Quarterly Reviews of Biophysics 50, e11. DOI 10.1017/S0033583517000099, https://doi.org/10.1017/S0033583517000099
  16. Artem K. Efremov, Ricksen S. Winardhi, Jie Yan (2017). Theoretical Methods for Studying DNA Structural Transitions under Applied Mechanical Constraints. Polymers 9, 74. DOI 10.3390/polym9020074, https://doi.org/10.3390/polym9020074
  17. Matteo Cristofalo, Daniel Kovari, Roberta Corti, Domenico Salerno, Valeria Cassina, David Dunlap, Francesco Mantegazza (2019). Nanomechanics of Diaminopurine-Substituted DNA. Biophysical Journal 116, 760–771. DOI 10.1016/j.bpj.2019.01.027, https://doi.org/10.1016/j.bpj.2019.01.027
  18. Xiao-Cong Zhao, Hang Fu, Lun Song, Ya-Jun Yang, Er-Chi Zhou, Guang-Xue Liu, Xue-Feng Chen, Zhuo Li, Wen-Qiang Wu, Xing-Hua Zhang (2019). S-DNA and RecA/RAD51-Mediated Strand Exchange in Vitro. Biochemistry 58, 2009–2016. DOI 10.1021/acs.biochem.8b01125, https://doi.org/10.1021/acs.biochem.8b01125
  19. Jack W. Shepherd, Robert J. Greenall, Matt I. J. Probert, Agnes Noy, Mark C. Leake (2020). The emergence of sequence-dependent structural motifs in stretched, torsionally constrained DNA. Nucleic Acids Research 48, 1748–1763. DOI 10.1093/nar/gkz1227, https://doi.org/10.1093/nar/gkz1227
  20. Dongni Yang, Wenzhao Liu, Xiangyu Deng, Wei Xie, Hu Chen, Zhensheng Zhong, Jie Ma (2020). GC-Content Dependence of Elastic and Overstretching Properties of DNA:RNA Hybrid Duplexes. Biophysical Journal 119, 852–861. DOI 10.1016/j.bpj.2020.06.034, https://doi.org/10.1016/j.bpj.2020.06.034
  21. Adam S. Backer, Graeme A. King, Andreas S. Biebricher, Jack W. Shepherd, Agnes Noy, Mark C. Leake, Iddo Heller, Gijs J. L. Wuite, Erwin J. G. Peterman (2021). Elucidating the Role of Topological Constraint on the Structure of Overstretched DNA Using Fluorescence Polarization Microscopy. The Journal of Physical Chemistry B 125, 8351–8361. DOI 10.1021/acs.jpcb.1c02708, https://doi.org/10.1021/acs.jpcb.1c02708
  22. Vinoth Sundar Rajan, Sune Levin, Micah J. McCauley, Mark C. Williams, Ioulia Rouzina, L. Marcus Wilhelmsson, Fredrik Westerlund (2025). Force-induced melting and S-DNA pathways for DNA overstretching exhibit distinct kinetics. Nucleic Acids Research 53, gkae1183. DOI 10.1093/nar/gkae1183, https://doi.org/10.1093/nar/gkae1183
  23. Kenichi Umeda, Shin'nosuke Yamanaka, Motonori Imamura, Fritz Nagae, Shingo Fukuda, Hiroki Watanabe, Takayuki Uchihashi, Shoji Takada, Toshio Ando (2026). Real-Time Sub-Molecular Visualization of DNA Folding Using a Hybrid High-Speed AFM and Optical Tweezers System. The Journal of Physical Chemistry Letters 17, 1182–1191. DOI 10.1021/acs.jpclett.5c03517, https://doi.org/10.1021/acs.jpclett.5c03517