Characterization of protein induced fluorescence enhancement (PIFE): A label-free protein assay with short distance sensitivity

Abstract

Single molecule FRET (Förster Resonance Energy Transfer) has been recently widely used for monitoring the interactions between a protein and a nucleic acid. It typically requires a site-specific labeling of the protein, which can be labor-intensive and inefficient. In addition, for common dye pairs, the distance sensitivity of FRET lies only between 3-7 nm. In this report, we report on a newly developed fluorescence assay termed Protein Induced-Fluorescence Enhancement (PIFE), which circumvents protein labeling and displays sharp detection sensitivity in short distance regions, overcoming the inherent limitations of FRET. The enhancement of fluorescence is based on the photophysical phenomenon where the intensity of a fluorophore increases upon the proximal binding of a protein. Using BamHI, Rig-I, and RecA as test protein systems, we present a systematic calibration and characterization of the PIFE technique. We demonstrate that this method enables detection under 4nm distance range with the resolution up to a single base pair. The high resolution and short distance sensitivity combined with the ability to bypass protein labeling makes this assay a readily accessible method to study any protein-nucleic acid activity which is an effective alternative and a complement to single molecule FRET.