When torsionally unconstrained double-stranded DNA is stretched, it elongates by about 70% at a nearly constant force of roughly 65-70 pN. Bongini, Lombardi and Bianco (2014) computed the atomistic free-energy profile of this B-to-S transition by umbrella-sampling molecular dynamics, reproducing several experimental thermodynamic quantities but overestimating the coexistence (plateau) force by about 50% (102 ± 9 pN vs ~70 pN experimentally) and predicting an S-state that is too short (0.52 vs 0.58 nm per base pair), with a helical repeat of 32 base pairs per turn, somewhat smaller than the 37.5 base pairs per turn reported from mechanical measurements (the source paper notes that 32 base pairs per turn agrees with the torque-measurement value, for which it gives no number). They attributed the discrepancy plausibly to overestimated base-stacking interactions in the AMBER parmBSC0 force field, and predicted that the plateau force should be lower for AT-rich sequences and the cooperativity index roughly proportional to GC content. These issues remain open: no atomistic free-energy study reproduces the experimental coexistence force and S-state geometry, systematic analysis of the sequence dependence of the B-to-S transition is still lacking, and the ~200-base-pair-scale simulation needed to test the sequence-based cooperativity mechanism has not been performed. The problem asks for a validated microscopic account of overstretching thermodynamics and its sequence dependence, with the quantitative benchmarks and controls stated explicitly.

Background

Double-stranded (ds) DNA in its canonical B-form extends from 0.34 nm to 0.58 nm per base pair when pulled from its ends, undergoing an overstretching transition at a nearly force-independent plateau of about 65-70 pN under physiological salt (Smith et al. 1996; Wenner et al. 2002 reviewed the salt dependence). The nature of the overstretched state has been contested for decades: candidate descriptions range from strand separation (force-induced melting, proceeding by peeling from nicks or free ends and by internal melting bubbles) to extended double-stranded forms collectively called S-DNA, with experiments showing the two coexist and compete (King et al. 2013; Zhang et al. 2013; Bosaeus et al. 2014). Under conditions that inhibit melting, overstretching proceeds without strand separation and behaves as a cooperative two-state reaction whose elementary units were measured to be about 22 base pairs on average (Bianco et al. 2011); the cooperativity index q denotes the number of base pairs that overstretch as one cooperative unit. Bongini, Lombardi and Bianco (2014) studied a 23-base-pair duplex with atomistic molecular dynamics (MD), obtaining the free-energy profile as a function of base-pair rise by umbrella sampling (a technique that biases the simulation along a reaction coordinate in overlapping windows and recombines them, here via the WHAM algorithm, into a single profile) with the AMBER parmBSC0 force field. Their profile is monotonic at zero force but two-state at high force, and it reproduces several measured quantities: the enthalpic and entropic contributions to the B-to-S free-energy difference (about 10 and 6 kB T per base pair, versus experimental 8.2 and 4.4), the transition-state extension, and the enthalpic part of the barrier. The mechanism they propose links the ~22-base-pair cooperativity unit to the average spacing between adenine/thymine (AT)-rich regions, where base pairing is disrupted in the simulated S state while the duplex is held together by non-native stacking, hydrophobic contacts and hydrogen bonds to the opposite backbone. However, the simulated coexistence force (102 ± 9 pN, defined as the force at which the B and S minima of the tilted free-energy profile are equal) exceeds the experimental ~70 pN at the same ionic strength by about 50%; earlier MD studies (Luan and Aksimentiev 2008; Roe and Chaka 2009) found similarly high values. The simulated S state is also too short (rise 0.52 instead of 0.58 nm per base pair), and its helical repeat of 32 base pairs per turn is somewhat smaller than the 37.5 base pairs per turn reported from mechanical measurements, although the source paper notes that 32 base pairs per turn agrees with the torque-measurement value (no number given in the source). The authors suspected an excessive weight of base-stacking interactions in parmBSC0 - the energetic difference between the two states is small enough that per-base-pair stacking errors translate into tens of pN in the coexistence force - and called for a more extensive analysis with different force fields. They also predicted, from the AT-rich-region mechanism, that the plateau force should be lower for AT-rich sequences and that q should be roughly proportional to GC content, awaiting tests on sequences with controlled base-pair composition; noted that testing the sequence-based cooperativity mechanism directly requires simulating ~200 base pairs, then unfeasible with explicit solvent; and observed in a control simulation that a zipper-like S-DNA candidate (each unpaired base hydrogen-bonded to and stacked between bases of the opposite strand, previously reported in simulations above 100 pN by Balaeff et al. 2011) was unstable in the range of forces they analysed. Here AT and GC denote the adenine-thymine and guanine-cytosine base pairs, kB T is thermal energy, and 'ionic strength' fixes the electrostatic screening of the negatively charged backbones.

Problem Statement

Can the DNA overstretching transition be given a quantitatively validated microscopic account, resolving the specific issues left open by the 2014 atomistic free-energy study of Bongini, Lombardi and Bianco? Concretely: (1) Determine whether the discrepancies between that simulation and experiment - a coexistence force of 102 ± 9 pN versus the experimental ~70 pN at matched ionic strength, an S-state rise of 0.52 versus 0.58 nm per base pair, and a helical repeat of 32 base pairs per turn versus the 37.5 base pairs per turn reported from mechanical measurements, which is the benchmark for the S-state helical repeat (the source paper notes 32 base pairs per turn agrees with the torque-measurement value, for which it gives no number, so that torque-derived value is non-quantitative context rather than a benchmark) - are force-field artifacts (for example overstabilized base stacking in parmBSC0), and produce an atomistic free-energy account of the B-to-S transition, at the ionic strengths where the experimental benchmarks exist, whose coexistence force, S-state geometry and enthalpic/entropic decomposition agree with experiment; this includes establishing whether the sequence-based cooperativity mechanism (cooperative units of ~22 base pairs set by the spacing of AT-rich regions) survives at the ~200-base-pair scale the 23-base-pair simulation could not address. (2) Determine experimentally, using sequences of controlled base-pair composition under conditions that suppress peeling and internal melting, whether the overstretching plateau force is lower for AT-rich sequences and whether the cooperativity index q scales roughly with GC content as the mechanism predicts. Parts (1) and (2) correspond to two independent open directions posed by the source paper - (1) its call for a more extensive analysis with different force fields, and (2) its experimentally verifiable predictions - and resolving either direction addresses its own open target.

References

  1. Bongini L., Lombardi V., Bianco P. (2014). The transition mechanism of DNA overstretching: a microscopic view using molecular dynamics. J. R. Soc. Interface 11, 20140399. DOI 10.1098/rsif.2014.0399. https://doi.org/10.1098/rsif.2014.0399
  2. Wenner J. R., Williams M. C., Rouzina I., Bloomfield V. A. (2002). Salt dependence of the elasticity and overstretching transition of single DNA molecules. Biophys. J. 82, 3160-3169. DOI 10.1016/S0006-3495(02)75658-0. https://doi.org/10.1016/S0006-3495(02)75658-0
  3. Bianco P., Bongini L., Melli L., Dolfi M., Lombardi V. (2011). PicoNewton-millisecond force steps reveal the transition kinetics and mechanism of the double-stranded DNA elongation. Biophys. J. 101, 866-874. DOI 10.1016/j.bpj.2011.06.039. https://doi.org/10.1016/j.bpj.2011.06.039
  4. Léger J. F., Romano G., Sarkar A., Robert J., Bourdieu L., Chatenay D., Marko J. F. (1999). Structural transitions of a twisted and stretched DNA molecule. Phys. Rev. Lett. 83, 1066-1069. DOI 10.1103/PhysRevLett.83.1066. https://doi.org/10.1103/PhysRevLett.83.1066
  5. Bryant Z., Stone M. D., Gore J., Smith S. B., Cozzarelli N. R., Bustamante C. (2003). Structural transitions and elasticity from torque measurements on DNA. Nature 424, 338-341. DOI 10.1038/nature01810. https://doi.org/10.1038/nature01810
  6. Luan B., Aksimentiev A. (2008). Strain softening in stretched DNA. Phys. Rev. Lett. 101, 118101. DOI 10.1103/PhysRevLett.101.118101. https://doi.org/10.1103/PhysRevLett.101.118101
  7. Roe D. R., Chaka A. M. (2009). Structural basis of pathway-dependent force profiles in stretched DNA. J. Phys. Chem. B 113, 15364-15371. DOI 10.1021/jp906749j. https://doi.org/10.1021/jp906749j
  8. Balaeff A., Craig S. L., Beratan D. N. (2011). B-DNA to zip-DNA: simulating a DNA transition to a novel structure with enhanced charge-transport characteristics. J. Phys. Chem. A 115, 9377-9391. DOI 10.1021/jp110871g. https://doi.org/10.1021/jp110871g
  9. King G. A., Gross P., Bockelmann U., Modesti M., Wuite G. J. L., Peterman E. J. G. (2013). Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching using fluorescence microscopy. Proc. Natl. Acad. Sci. USA 110, 3859-3864. DOI 10.1073/pnas.1213676110. https://doi.org/10.1073/pnas.1213676110
  10. Zhang X., Chen H., Le S., Rouzina I., Doyle P. S., 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. USA 110, 3865-3870. DOI 10.1073/pnas.1213740110. https://doi.org/10.1073/pnas.1213740110
  11. Bosaeus N., El-Sagheer A. H., Brown T., Akerman B., Norden B. (2014). Force-induced melting of DNA - evidence for peeling and internal melting from force spectra on short synthetic duplex sequences. Nucleic Acids Res. 42, 8083-8091. DOI 10.1093/nar/gku441. https://doi.org/10.1093/nar/gku441
  12. Bongini L., Melli L., Lombardi V., Bianco P. (2014). Transient kinetics measured with force steps discriminate between double-stranded DNA elongation and melting and define the reaction energetics. Nucleic Acids Res. 42, 3436-3449. DOI 10.1093/nar/gkt1297. https://doi.org/10.1093/nar/gkt1297
  13. Marin-Gonzalez A., Vilhena J. G., Perez R., Moreno-Herrero F. (2017). Understanding the mechanical response of double-stranded DNA and RNA under constant stretching forces using all-atom molecular dynamics. Proc. Natl. Acad. Sci. USA 114, 7049-7054. DOI 10.1073/pnas.1705642114. https://doi.org/10.1073/pnas.1705642114
  14. Garai A., Mogurampelly S., Bag S., Maiti P. K. (2017). Overstretching of B-DNA with various pulling protocols: Appearance of structural polymorphism and S-DNA. J. Chem. Phys. 147, 225102. DOI 10.1063/1.4991862. https://doi.org/10.1063/1.4991862
  15. Bosaeus N., Reymer A., Beke-Somfai T., Brown T., Takahashi M., Wittung-Stafshede P., Rocha S., Norden B. (2017). A stretched conformation of DNA with a biological role? Q. Rev. Biophys. 50, e11. DOI 10.1017/S0033583517000099. https://doi.org/10.1017/S0033583517000099
  16. Schakenraad K., Biebricher A. S., Sebregts M., Ten Bensel B., Peterman E. J. G., Wuite G. J. L., Heller I., Storm C., van der Schoot P. (2017). Hyperstretching DNA. Nat. Commun. 8, 2197. DOI 10.1038/s41467-017-02396-1. https://doi.org/10.1038/s41467-017-02396-1
  17. Bongini L., Pongor C., Falorsi G., Pertici I., Kellermayer M., Lombardi V., Bianco P. (2016). An AT-barrier mechanically controls DNA reannealing under tension. Nucleic Acids Res. 44, 7954-7962. DOI 10.1093/nar/gkw604. https://doi.org/10.1093/nar/gkw604
  18. Kovari D. T., Cristofalo M., Corti R., Salerno D., Cassina V., Lee Y., Malla G., Finzi L., Mantegazza F., Dunlap D. (2018). Experimentally motivated sequence-dependent models of melting and overstretching for diaminopurine-substituted DNA. Biophys. J. 114 (Biophysical Society Meeting Abstracts), 88a. DOI 10.1016/j.bpj.2017.11.524. https://doi.org/10.1016/j.bpj.2017.11.524
  19. Cristofalo M., Kovari D., Corti R., Salerno D., Cassina V., Dunlap D., Mantegazza F. (2019). Nanomechanics of diaminopurine-substituted DNA. Biophys. J. 116, 760-771. DOI 10.1016/j.bpj.2019.01.027. https://doi.org/10.1016/j.bpj.2019.01.027
  20. Melkonyan L., Bercy M., Bizebard T., Bockelmann U. (2019). Overstretching double-stranded RNA, double-stranded DNA, and RNA-DNA duplexes. Biophys. J. 117, 509-519. DOI 10.1016/j.bpj.2019.07.003. https://doi.org/10.1016/j.bpj.2019.07.003
  21. Yang D., Liu W., Deng X., Xie W., Chen H., Zhong Z., Ma J. (2020). GC-content dependence of elastic and overstretching properties of DNA:RNA hybrid duplexes. Biophys. J. 119, 852-861. DOI 10.1016/j.bpj.2020.06.034. https://doi.org/10.1016/j.bpj.2020.06.034
  22. Sundar Rajan V., Levin S., McCauley M. J., Williams M. C., Rouzina I., Wilhelmsson L. M., Westerlund F. (2025). Force-induced melting and S-DNA pathways for DNA overstretching exhibit distinct kinetics. Nucleic Acids Res. 53, gkae1183. DOI 10.1093/nar/gkae1183. https://doi.org/10.1093/nar/gkae1183
  23. Afanasyev A. Y., Onufriev A. V. (2025). Multiplateau force-extension curves of long double-stranded DNA molecules. ACS Omega 10, 14816-14825. DOI 10.1021/acsomega.4c09605. https://doi.org/10.1021/acsomega.4c09605
  24. Burman M., Noy A. (2025). Atomic description of the reciprocal action between supercoils and melting bubbles on linear DNA. Phys. Rev. Lett. 134, 038403. DOI 10.1103/PhysRevLett.134.038403. https://doi.org/10.1103/PhysRevLett.134.038403
  25. Clausen-Schaumann H., Rief M., Tolksdorf C., Gaub H. E. (2000). Mechanical stability of single DNA molecules. Biophys. J. 78, 1997-2007. DOI 10.1016/S0006-3495(00)76747-6. https://doi.org/10.1016/S0006-3495(00)76747-6