DNA’s extension increases by 70% in a narrow force window at 65 pN, a phenomena called 'over stretching'. The state of the DNA after overstretching has been controversial since the discovery of overstretching in 1996. One group of scientists advocate for S-DNA, where the rise per base pair and the tilt is significantly altered. Another group of scientists argue that this is completely wrong, and that overstretching DNA is really a force-induced melting.
Given this ongoing uncertainty, we sought to directly test if the generation of single stranded (ss) DNA via a peeling mechanism – the leading mechanism for overstretching DNA (PNAS, 2009, 106, 18231) – is necessary for overstretching DNA. However, to date, limitations in substrates have led to a discontinuous transition between stretching DNA that has no free ends (Fo = 110 pN) and DNA with nicks or free ends (Fo = 65 pN) because assays containing no free ends were both topologically closed and torsionally constrained. We developed a new topologically closed but rotationally unconstrained assay by binding DNA at one end to a surface by both strands and the other end to a bead via an internal biotin embedded in a 5-nucleotide loop.
In summary, DNA in the topologically closed assay overstretched at the canonical 65 pN, but lackedhysteresis. Hence, ssDNA generation from nicks or free ends – the leading mechanism for overstretching DNA is not a compulsory step in overstretching. Rather, such peeling is the primary cause of hysteresis in the force-extension curve. We hope that these new findings on the mechanism of overstretching DNA will narrow the range of the ongoing debate about overstretched DNA.