When double-stranded DNA is pulled to about 65 pN it elongates by roughly 70% (the overstretching transition). A landmark 2009 multicolor single-molecule fluorescence study concluded that overstretching is force-induced melting and posed four linked questions: whether torsionally constrained overstretched DNA consists of two melted single strands wrapped around each other or an intact base-paired form; what the kinetics and thermally activated barriers are for melting the final hundreds of base pairs, which remain double-stranded out of equilibrium; what the smallest, sequence-dependent nucleation events are; and what the structural organization of fully overstretched, end-constrained DNA is. Follow-up work between 2011 and 2021 dissolved the central dichotomy: overstretched DNA is a heterogeneous, topology-, force-, sequence-, and salt-dependent coexistence of peeled single strands, internal melting bubbles, a base-paired elongated form (S-DNA), and an overwound form (P-DNA); melting bubbles nucleate in AT-rich regions and S-DNA base pairs are inclined by about 54 degrees. What remains open is the completion of this characterization: the local helical parameters and strand topology of S-DNA and P-DNA under defined constraints, the sub-30-base-pair nucleation events, and quantitative maps of melting barriers and equilibration times versus force, linking number, sequence, and salt — with a 2025 study still describing the structure, energetics, and kinetics of S-DNA as unresolved.

Background

Double-stranded (ds) DNA in its physiological B-form is a right-handed double helix of about 10.5 base pairs (bp) per turn. In optical-tweezers experiments, in which a single DNA molecule is tethered between micron-scale beads and stretched while its force-extension curve is recorded, torsionally relaxed dsDNA undergoes a sharp structural transition at about 65 pN, elongating the molecule by roughly 70% (Smith, Cui and Bustamante, 1996). This is called overstretching. Since its discovery, two competing pictures have been debated: a cooperative transition to an elongated but intact base-paired duplex (called S-DNA), versus force-induced melting, in which the hydrogen bonds between base pairs break and single-stranded (ss) DNA is formed, analogous to thermal melting (Cocco et al., 2004).

Several structures are distinguished in this debate. Strand peeling (unpeeling) means that one strand detaches from the other starting at a free end or a nick (a break in one strand's phosphodiester backbone), so that one strand carries the tension while the other coils. Melting bubbles are short internal regions in which the two strands separate while both termini remain attached, so both strands share the tension. S-DNA is a hypothetical elongated duplex in which base pairing is maintained but the base pairs are strongly inclined; P-DNA is an overwound form, predicted to have about 2.5 bp per turn, in which the two backbones wrap tightly around each other and bases flip outward. Torsional constraint means the total linking number Lk=Tw+WrLk = Tw + Wr (the sum of the helical winding of the strands and the coiling of the helix axis) is fixed because both strands are anchored at both ends; such DNA overstretches only near 110 pN instead of 65 pN, since it cannot release tension by unwinding. Key experimental readouts are fluorescent probes: replication protein A (RPA) and single-strand-binding protein, which bind specifically to ssDNA and thereby mark melted or peeled regions, and intercalating dyes, which insert between base pairs and thereby mark double-stranded DNA. Because the ssDNA-binding proteins bind cooperatively, they mark single-stranded regions only down to roughly 30 base pairs, which sets the practical detection limit for small melting bubbles.

Van Mameren et al. (2009) combined optical tweezers, microfluidics and multicolor fluorescence imaging and concluded that overstretching is a gradual conversion of dsDNA to ssDNA regardless of attachment geometry, initiating at nicks and free ends, and, in intact (torsionally constrained) molecules, occurring throughout the molecule near 110 pN. They proposed that this 110-pN state consists of two melted ssDNA strands wrapped around each other, and speculated that an earlier reported structure of about one helical turn per 35 bases, obtained when underwound torsionally constrained DNA was stretched to about 50 pN (Leger et al., 1999), might also be melted wrapped strands. They noted that melting the last hundreds of base pairs is out of equilibrium on experimental timescales, that their imaging resolution (about 250 nm, roughly 750 bp) could not resolve small internal bubbles, and that the GC-rich half of lambda DNA (the roughly 48,500-base-pair genome of bacteriophage lambda; GC-rich means enriched in guanine-cytosine base pairs, AT-rich in adenine-thymine pairs) appeared to melt last.

Subsequent work transformed this picture. Fu et al. (2011) showed that S-DNA is stable even in nicked molecules under physiological salt and that GC-rich islands select the transition pathway. Zhang et al. (2012) distinguished two overstretched structures thermodynamically in end-opened versus end-closed constructs. Bosaeus et al. (2012) proved with designed 60-64 bp duplexes that GC-rich DNA undergoes a reversible transition to a base-paired overstretched form (51% extension), whereas AT-rich DNA denatures. King et al. (2013) and Zhang et al. (2013), in back-to-back studies, showed that all three structures - peeled ssDNA, melting bubbles and S-DNA - coexist, with the balance governed by DNA topology, local sequence stability and ionic conditions; melting bubbles were found to nucleate in AT-rich regions. King et al. (2016) then performed exactly the experiment requested in 2009, combining fluorescence imaging with force and torsional control: in torsionally constrained DNA, base-pair melting and helical unwinding occur concomitantly above about 50 pN (local changes in linking number are absorbed by the rest of the duplex), and above about 110 pN an overwound structure, likely P-DNA, forms alongside underwound regions. Backer et al. (2019) used polarization microscopy of intercalating dyes to show that S-DNA base pairs are inclined by about 54 degrees relative to the DNA axis, the first direct structural constraint on S-DNA, and Backer et al. (2021) showed that torsional constraint and negative supercoiling substantially alter intercalator orientations, indicating P-DNA formation. Kinetic work (Bongini et al., 2014; Zhang et al., 2014; Sundar Rajan et al., 2025) established that the B-to-S transition is fast, reversible and largely non-hysteretic, whereas melting is hysteretic and much slower. Nevertheless, a 2025 study states that the structure, energetics, kinetics and biological role of S-DNA remain obscure, and no atomic-level or full helical-parameter description of the overstretched states exists.

Problem Statement

The 2009 question of whether torsionally constrained overstretched DNA is melted wrapped ssDNA or an intact base-paired form has been answered - it is a heterogeneous, constraint-dependent mixture - and the surviving problem is to complete the structural and kinetic characterization that this answer now demands. Specifically: (1) determine the structural organization of DNA overstretched under defined topological constraints (torsionally constrained, i.e. with linking number fixed; topologically closed but rotationally free, i.e. strand peeling blocked but twist still able to relax; and end-opened, i.e. strands free to peel from free ends), including the local helical parameters and strand topology of the S-DNA and P-DNA domains, the spatial arrangement and coexistence ratios of melted, base-paired and overwound regions along the molecule, and the smallest melting-bubble nucleation events below the roughly 30 bp fluorescence detection limit; and (2) quantify the kinetics and thermally activated barriers of force-induced DNA melting - in particular the out-of-equilibrium terminal-segment melting that leaves the final hundreds of base pairs double-stranded at forces beyond the transition - and establish the conditions of force, linking number, sequence and ionic strength that determine equilibration times. Each of the two parts, and each enumerated result within them, is a mandatory deliverable rather than an illustrative example: establishing any single sub-result is a partial contribution to the objective, not a resolution of the task. Both parts serve one objective: a complete, experimentally grounded structural and kinetic account of the overstretched state of DNA under tension, replacing the qualitative 2009 picture and its later qualitative refinements with quantitative structure determination and rate and barrier maps. Answers may combine single-molecule experiments (force spectroscopy with fluorescence, polarization or high-speed atomic force microscopy (AFM) readout, under torsional control) with validated structural or kinetic modeling.

References

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