Aim: 

We use a dual-laser optical-tweezers (DLOT) to study the highly cooperative conformational transition in the molecule of DNA, where the natural B-DNA is converted into a new overstretched conformation called S-DNA (Bensimon et al., Phys. Rev. Lett.74, 4754, 1995; Cluzel et al., Science271, 792, 1996; Smith et al., Science271, 795, 1996).

Methods: 

Single molecules of double stranded l-phage DNA are stretched at 27 °C with the DLOT either with ramp length changes or with force step staircases. The force transducer was calibrated against the viscous drag on a bead by using Stokes' law. The biotinylated ends of a double stranded DNA molecule are tethered to streptavidin coated beads (3.18 mm diameter), one trapped in the optical tweezers and the other attached to the pipette carried by the piezo manipulator. The microchamber solution contains 150 mM NaCI, 10 mM tris-HCl, 1 mM EDTA, pH 8.0.

Results: 

When the DNA molecule is stretched in length clamp mode, it shows the previously described highly cooperative overstretching transition at ~60 pN, attributed to unwinding from the B-form to the 1.7 times longer S-form. Upon subsequent relaxation, the force drops more steeply, exhibiting a marked hysteresis attributed to the melting of one strand in the overstretched molecule (Smith et al., 1996). Stretching the same molecule in force clamp mode with a staircase of force steps of 5 pN at 5 s intervals allows to determine the amount of DNA elongation (L) as a function of the clamped force (F). The L-F relation is a sigmoid and the fitting with Hill equation gives n (the Hill coefficient expressing the steepness of the relation) ~20 and k (the value of F at which L attains 0.5 the maximum value) ~60 pN.

Conclusions: 

Stretching DNA with our force step protocol gives evidence that DNA does not unwind as single bps, but as a sequence of blocks of bps, with average block size of 20 bps.

Supported by MiUR, Ente Cassa di Risparmio di Firenze (2005. 1245) and ITB-CNR (Milano).