Single-molecule visualization and manipulation of protein and DNA interactions and dynamics, on nanoscale dimensions

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The overarching objective of my research program is to visualize, quantitatively analyze and understand protein-DNA and protein-protein interactions, which play key biophysical roles in regulating cellular processes. I am fascinated by the interplay between the dynamic, structural, and genetic properties of biopolymers such as protein and DNA molecules, which advancements in visualization and nano manipulation tools, as well as theoretical treatments of confined molecules, are allowing us to understand in further detail. This Discovery Grant will enable my group to leverage our single-molecule microscopy tools to explore open questions about the behavior of protein and DNA systems on nanoscale dimensions; questions which are inaccessible to other methods, and which have international research interest and impact.******My Research Program will pursue 2 Discovery Projects, with an interdisciplinary team. Project 1 will visualize the interactions between "supercoiled" DNA and "probe molecules" which are designed to bind to specific "target sites". These studies will address open questions about how the conformations of DNA can influence the unwinding of the target sites, as well as consequent binding events to these sites, by watching the events occur in real time. These measurements will test biophysical theories which are being developed to describe a wide range "topology-mediated processes" in DNA, motivated by seeking to understand gene regulation in living cells. Project 2 will visualize the behavior of protein systems on nanoscale dimensions; in particular we will study how protein-protein interactions regulate the formation of "liquid droplets", which in turn serve specific functionalities inside cells. These projects will leverage perspectives of our collaborators in theory for modeling, in biochemistry/molecular biology for sample preparation, and atomic force microscopy for high-resolution visualization.******Our unique microscopy devices enable crisp movies of molecular interactions and dynamics to be acquired, under known confinement and solution conditions, by gently "squeezing" individual molecules into custom chambers. Our techniques enable us to detect weak and slow interaction events and to take continuous movies of molecular trajectories over tens of seconds to minutes. By confining molecules to the focal plane, our approach is “handle-free”, allowing the molecules to explore their full configurational space while interacting with each other; this means we can discern the influence of DNA conformation upon protein-binding events, for example. We will design molecular systems to systematically increase in complexity, approaching conditions in the "cellular milieu". Our studies of biophysical mechanisms, enabled by new physical tools in combination with new biophysical data and modeling, will provide a foundation for advancements in biomedicine.