Nanopore Sensing of DNA-Histone Complexes on Nucleosome Arrays.

The location of nucleosomes in DNA and their structural stability are critical in regulating DNA compaction, site accessibility, and epigenetic gene regulation. Here, we combine the nanopore platform-based fast and label-free single-molecule detection technique with a voltage-dependent force rupture assay to detect distinct structures on nucleosomal arrays and then to induce breakdown of individual nucleosome complexes. Specifically, we demonstrate direct measurement of distinct nucleosome structures present on individual 12-mer arrays. A detailed event analysis showed that nucleosomes are present as a combination of complete and partial structures, during translocation through the pore. By comparing with the voltage-dependent translocation of the mononucleosomes, we find that the partial nucleosomes result from voltage-dependent structural disintegration of nucleosomes. High signal-to-noise detection of heterogeneous levels in translocation of 12-mer array molecules quantifies the heterogeneity and nucleosomal substructure sizes on the arrays. These results facilitate the understanding of electrostatic interactions responsible for the integrity of the nucleosome structure and possible mechanisms of its unraveling by chromatin remodeling enzymes. This study also has potential applications in chromatin profiling.

Temperature dependent in vitro binding and release of target DNA by Cas9 enzyme.

The CRISPR-associated protein 9 (Cas9) system has proven to be a powerful technology for genome editing in a wide variety of in vivo and in vitro applications. CRISPR-Cas9, when loaded with the guide RNA, cleaves the DNA at the target position as recognized by the guide RNA sequence. For successful application of this technology, it is important to study the biophysical parameters affecting its function. Temperature dependence of the Cas9 binding as well as energetics of product release after cleavage has not been well reported in the literature. In this work, we study the binding properties of Cas9 enzyme to the sequence specific target DNA at a range of temperatures and, surprisingly, find that the Cas9 enzyme, in our study, can find and bind its target DNA with 90 ± 20% efficiency at temperatures as low as 4 °C. Further, we show that the cleaved DNA products remain bound to the Cas9 enzyme strongly and is released from the enzyme only at higher temperatures. Using the gel shift assays, we quantify the rate of Cas9 binding to target DNA to be 0.8 ± 0.2 min-1 at 37 °C. We also tested denaturant (SDS) dependent release of cleaved product which showed a similar release pattern with a dissociation constant of 0.23 ± 0.04 mM. Our results of heat and denaturant dependence on Cas9-DNA binding and release mechanics will provide valuable insights for developing temperature dependent applications of the CRISPR-Cas9 technology.