Structure and Dynamics of Supramolecular Systems one Molecule/Particle at a Time

Over the past years, the possibility of visualizing single fluorescent molecules with exquisite spatial and temporal resolution has given rise to the field of Single Molecule Spectroscopy (SMS). The technique has enabled establishing new paradigms in biophysics and chemical biology by revealing unique information otherwise hidden in traditional ensemble measurements. In this work, we apply SMS methodologies to address problems in the field of chemistry and self-assembled structures, specifically to sense small molecules bound to a lipid membrane, to unravel exciton migration in conjugated polyelectrolytes, to study protein-RNA interactions and to characterize DNA nanotubes in solution.;We also present single molecule Förster resonance energy transfer (SM-FRET) studies, on the dynamics of the non-structural RNA-dependent RNA polymerases (RdRp) 5B in HCV polymerase-substrate binding process. Our SMS-FRET work allowed us to obtain a direct visualization of both the positioning and dynamics of NS5B in complex with its RNA template and to study the mechanism of a non-nucleoside analogue inhibitor. SM-FRET studies on RNA-protein complexes revealed protein sliding dynamics occurring on the millisecond time scale. Sliding of NS5B provides a plausible mechanism that facilitates formation of a productive complex. A non-nucleoside inhibitor is observed to compromise binding of NS5B to the template.;We further discuss the use of fluorescence microscopy to characterize the structure and dynamics of single DNA nanotubes with different architectures. Our single molecule/particle fluorescence studies show that DNA-nanotubes exist in solution as a rigid rod with an average length of 10 μm. The selective opening of these DNA-nanotubes with specific DNA strands results in a floppy/collapsed structure.;Altogether, our single molecule experiments have provided a wealth of information and improved our understanding of supramolecular systems in complex dynamic interactions. These studies are at the heart of the fields of chemistry and biology.;We discuss here the preparation and characterization of a liposome beacon platform for sensing small molecules. This platform relies on the interaction of conjugated polyelectrolytes with lipid membranes. The effect different lipids have on the photophysical properties of the polymer are discussed. A molecular-level visualization of liposomes via Cryo-TEM combined with single molecule spectroscopy studies on conjugated polyelectrolytes (MPS-PPV) has enabled us to directly correlate the polymer conformation with its spectroscopic features. In aqueous solution, MPS-PPV adopts a collapsed-chain conformation leading to efficient energy migration over multiple chromophore units. These results correlate with the amplified sensing potential reported for MPS-PPV. When confined within neutral vesicles, single MPS-PPV molecules are shown to adopt an extended conformation upon insertion in the lipid bilayer, characterized by poor energy migration.