Single-Molecule Imaging With One Color Fluorescence

Single-molecule fluorescence imaging is a powerful tool that enables real-time observation of DNA-protein or RNA-protein interactions with a nanometer precision. Here, we provide a detailed procedure for a previously developed single-molecule fluorescence method, termed "single-molecule protein-induced fluorescence enhancement" (smPIFE). While smFRET (Förster resonance energy transfer) requires both donor and acceptor, protein-induced fluorescence enhancement (PIFE) employs a single dye and measures the increase in fluorescence intensity induced by protein binding near the dye. PIFE displays distance sensitivity within 0-4nm, making it a powerful complementary or alternative tool to FRET method. In this chapter, we will discuss the various ways that PIFE has been utilized to study protein-nucleic acid interactions.