The Photophysics of Pife and other Photophysical Process that Affect Single-Molecule Dyes

Abstract

Single-molecule techniques have transformed research in molecular biochemistry and biophysics by allowing scientists to investigate the dynamics and heterogeneity of individual biomolecules. In this talk I will discuss our work characterizing how the spectroscopic and photophysical properties of common fluorescent probes used in single molecule experiments are affected by conjugation to biopolymers. A careful characterization of the photophysical properties of these probes is not only critical for the appropriate design and interpretation of fluorescence experiments, but also enables the discovery of new uses of known fluorescent probes. An example of this is the use of the cyanine Cy3 as a probe for “protein proximity”. The strategy, termed PIFE for protein induced fluorescence enhancement, has proven to be very useful in the investigation of the single-molecule dynamics of wild-type (i.e. unlabeled) proteins that translocate or diffuse on DNA or RNA. We have elucidated the photophysical mechanism that leads to protein-enhanced fluorescence emission of Cy3 when in close proximity to a protein. Understanding the photophysics behind PIFE will enable the development of other probes that can be used in similar experiments in other regions of the spectrum (e.g. in the red).