Probing DNA Stiffness with Magnetic Tweezers

Abstract

The exceptional stiffness of DNA is routinely attributed to base stacking and repulsion between neighboring, negatively charged phosphates. Furthermore, well established biochemistry and recent single molecule experiments show that small, charged molecules and intercalating agents can dramatically alter the pitch and twist of the double helix. It is very likely that cells and macromolecular complexes have evolved to influence these phenomena in order to alter the flexibility of DNA to efficiently catalyze DNA transactions. In order to gain insight into how cells manipulate DNA efficiently, a quantitative understanding of DNA elasticity is necessary. Using magnetic tweezers to twist and stretch single DNA molecules, experiments were performed to probe the parameters of DNA stiffness. Although this technique is exquisitely sensitive and permits broad exploration of twist versus extension data, accurate models with which to interpret the stiffness parameters are critical. In particular, the knee point of the DNA extension versus twist curve, which is sensitive to both the bending and torsional rigidity of the molecule, is a signature, which if modeled accurately might give insight into how base stacking and electrostatics contribute to DNA stiffness. Our experiments showed that diaminopurine substitution for adenine, which adds an additional hydrogen bond to AT base pairs, stiffens DNA by about 50% without significantly changing the knee point. Instead adding low molecular weight polycations such as spermine or spermidine to the solution appeared to soften DNA and promote plectoneme formation at lower values of torsion. Thus base pair stability and, implicitly, stacking seem to have affected only the DNA elasticity while charge neutralization also favored the conversion of excess twist into writhe.