Research Projects

Our proposed research project aims to gain a comprehensive understanding of the DNA Holliday junction (HJ) conformational dynamics and branch migration kinetics at single molecule level in real-time with potential application as a biosensor against cancer. Our research uses state-of-the-art optical microscopy techniques such as single molecule fluorescence resonance energy transfer (smFRET), super-resolution imaging, and fluorescence lifetime correlation spectroscopy (FLCS). DNA Holliday junctions are critical intermediate structures that form when four DNA double strands intersect during homologous genetic recombination. Depending on the DNA sequence, branch migration may be equally likely in any of the four branches, determining the efficiency and fidelity of the recombination process. Therefore, understanding the molecular mechanisms of the stochastic conformational dynamic process coupled with fast branch migration is critical. During conformational fluctuation, HJ can go from a fully open to a fully closed state through a complex network of intermediate conformational states depending on conditions. How stochastic time series of conformational dynamics influence the branch migration to perform the exact amount of genetic recombination is still unresolved. Several complex kinetic models based on Hidden Markov Models (HMM) analysis have been proposed, predicting the kinetics of hidden intermediate states based on the observable states (fully open or closed). The fundamental assumption of HMM is that fully open or fully closed structures of DNA HJ at t = 0 must be influenced by the hidden conformations, and the future conformations will depend on the present, not the past states. However, a fundamental problem of these models is the lack of direct observation of hidden intermediate states, which may directly influence the observable structures. Therefore, our hypothesis is that during conformational dynamics coupled with directional branch migration, DNA HJ goes through countless intermediate stochastic states within observable structures that depend on various thermodynamic parameters. A small deviation of base-pair, pH, charge density, and thermodynamic parameters may lead to the formation of other hidden states within the known gaussian structural distribution. Our proposed research will use sophisticated microscopy techniques and statistical analysis to determine a comprehensive molecular mechanism of conformational dynamics and branch migration of DNA HJ on a fast time-scale. We also propose developing a biosensor based on DNA HJ. Our proposed research will directly “visualize” DNA HJ conformational dynamics coupled with directional branch migration kinetics. Ultimately, it may help solve long-stating fundamental questions, build new knowledge, and aid in early cancer diagnosis.

smFRET, Super-resolution Imaging, FLCS, DNA Holliday Junction, Conformation Dynamics, Biosensor