Torque Measurements during the Spontaneous Unbraiding of DNA Molecules Showed Large Fluctuations Attributable to the Formation of Stable DNA-DNA Interactions

Abstract

Magnetic tweezers dual-molecule braiding assays provide an adequate framework for studying the mechanical aspects underlying the process of winding two DNA molecules around each other. In its typical implementation however, this technique do not allow direct measurement of torque because the torque exerted by the magnetic traps (∼103 pN nm) is at least one order of magnitude larger than the restoring torque of DNA (∼ 102 pN nm). In previous work, our laboratory showed that enough elastic energy could be stored in a pair of braided DNA molecules to perform mechanical work by rotating a microscopic object in absence of manipulation. Here we have exploited that unique property of DNA to perform a real-time study of braided DNA dynamics. In our experiments, torsionally unconstrained lambda DNA molecules were bound to a surface by one end and to a microscopic dumbbell at the other. A magnetic tweezers apparatus was employed to stretch and braid the molecules. Upon creating the braid, the magnet was removed to trigger the spontaneous unbraiding of DNA. The rotational dynamics of the dumbbell was followed in real-time. Hydrodynamic equations were employed to relate the dynamical information of the dumbbells to torque values. Our most important finding is that the unbraiding process occurs in a discontinuous manner. The estimated magnitudes of torque were found to fluctuate between an upper (∼ 102 pN nm) and a lower bound (∼ 101 pN nm) value along the entire process. The magnitudes of the fluctuations were found to be independent of the amount of times that the molecules crossed each other. No different was found for braids of opposite handedness. We attributed these fluctuations to the formation/rupture of stable DNA interactions.