Convex lens induced confinement : a novel study of weak and slow DNA & protein interactions

Abstract

The newly developed single molecule microscopy method of flow cell-convex lens induced confinement (FC-CLIC) allows for the previously unobtainable probing of slow and weak DNA and protein interactions. Such measurements are made possible by CLIC's simultaneously improved signal-to-background ratio, observation time, observation volume, and permissible molecule concentration. The 10, 000 increase in the observation time is demonstrated with the protein-protein interaction between actin filaments and formin. CLIC is used to generate data of the binding of a HMG protein to double-stranded DNA with a sufficient signal-to-background over long enough times for efficient particle tracking. The protein-DNA complex's diffusion coefficient is also extracted and compared with past measurements to successfully verify the accuracy of CLIC's measurements. Finally, the application of fluorescence cross correlation spectroscopy (FCCS) to the conformation fluctuations of DNA hairpins via Fdrester resonance energy transfer (FRET) is demonstrated. Progress is made towards the future use of FCCS and CLIC to determine the hairpin conformational reaction rates. The unimolecular hairpin is the preliminary molecular system for the study of electrostatic interactions in the alignment of DNA fragments, a new theory for the alignment of homologous chromosomes.