Stretching DNA with optical tweezers

Abstract

Force-extension (F-x) relationships were measured for single molecules of DNA under a variety of buffer conditions, using an optical trapping interferometer modified to incorporate feedback control. One end of a single DNA molecule was fixed to a coverglass surface by means of a stalled RNA polymerase complex. The other end was linked to a microscopic bead, which was captured and held in an optical trap. The DNA was subsequently stretched by moving the coverglass with respect to the trap using a piezo-driven stage, while the position of the bead was recorded at nanometer-scale resolution. An electronic feedback circuit was activated to prevent bead movement beyond a preset clamping point by modulating the light intensity, altering the trap stiffness dynamically. This arrangement permits rapid determination of the F- x relationship for individual DNA molecules as short as ~1 μm with unprecedented accuracy, subjected to both low (~0.1 pN) and high (~50 pN) loads: complete data sets are acquired in under a minute. Experimental F-x relationships were fit over much of their range by entropic elasticity theories based on worm-like chain models. Fits yielded a persistence length, L(P), of ~47 nm in a buffer containing 10 mM Na1. Multivalent cations, such as Mg2+ or spermidine3+, reduced L(P) to ~40 nm. Although multivalent ions shield most of the negative charges on the DNA backbone, they did not further reduce L(P) significantly, suggesting that the intrinsic persistence length remains close to 40 nm. An elasticity theory incorporating both enthalpic and entropic contributions to stiffness fit the experimental results extremely well throughout the full range of extensions and returned an elastic modulus of ~1100 pN.