The organization of eukaryotic DNA into nucleosomes and chromatin involves dynamic structural changes. These dynamics are also relevant to regulate genome processing, including transcription, replication, and DNA repair. However, there is a lack of methodologies that probe structure and structural changes over mesoscopic (10-100nm) length scales within chromatin. We have designed, constructed, and implemented a DNA-based nanocaliper that probes this mesoscopic length scale to address these challenges. Our nanocalipers consist of 2 70nm rigid arms each made up of 18 helices of double stranded DNA (dsDNA), connected by 6 sets of single stranded DNA (ssDNA) at one end. The nanocaliper has the appearance of a hinge joint. The nanocaliper design and its ability to fluctuate between 0-120o allows for end-to-end distance measurements on the tens of nanometers length scale.
We developed an approach for integrating nucleosomes into our nanocaliper at two attachment points with over 50% efficiency. We focused on attaching a strand of DNA containing a nucleosome consensus sequence to the ends of the two nanocaliper arms, so the hinge angle acts as a readout of nucleosome end-to-end distance. We found that the nanocaliper angle is a sensitive measure of nucleosome disassembly and can read out transcription factor binding to its target site within the nucleosome. Interestingly, the nanocaliper not only detects transcription activator Gal4-VP16 binding but also significantly increases the probability of Gal4-VP16 occupancy at its respective site by contributing to nucleosome unwrapping. This suggests that our DNA nanocalipers can serve as a tool to readout biologically relevant conformational changes and to manipulate nucleosomes to test their function under different physical constraints.
We have also developed a model using this data to characterize how individual nucleosomes adopt unwrapped conformations. We find that this model demonstrates concomitant nucleosome unwrapping and is further validated by hexasome unwrapping experiments. We can use this model to study trends in the dynamics of nucleosomes, chromatin, and regulatory processes involving nucleosome unwrapping.
This project provides a foundation for future mesoscale studies of nucleosome and chromatin structural dynamics, an area that has been largely unexplored. More broadly, mesoscale studies of nucleosomes provide insight into molecular events that lead to misregulated oncogenes and tumorigenesis. The nanocaliper construct can be further scaled up to probe larger biomolecular complexes such as nucleosome arrays. Current work focuses on developing a caliper variation that is more compatible for nucleosome arrays in physiological conditions. Ultimately, these tools could be implemented inside cells to directly monitor or manipulate local chromatin structure and dynamics in single living cells.