Single Chromatin Fibre Assembly Using Optical Tweezers

Here we observe the formation of a single chromatin fibre using optical tweezers. A single -DNA molecule was suspended between two micron-sized beads, one held by a micropipette and the other in an optical trap. The constrained DNA molecule was incubated with Xenopus laevis egg extract in order to reconstitute a single chromatin fibre. An eight-fold compaction of the DNA molecule was observed in real-time. The compaction kinetics were found to be strongly dependent upon the tension applied to the DNA molecule. We incorporated the analysis of Brownian motion to accurately determine the tension throughout the compaction process. At forces exceeding 10 pN complete inhibition of compaction was observed for the time scale of the experiment. We have previously shown that stretching of a reconstituted chromatin fibre results in discrete and quantized structural opening events that we can attribute to the unravelling of single nucleosomes. Assembly kinetics therefore provide insight into rates of nucleosome formation and we demonstrate the possibility of probing these kinetics under different experimental conditions