Indexed on: 25 Oct '16Published on: 10 Oct '16Published in: Methods in enzymology
Publication date: Available online 7 October 2016 Source:Methods in Enzymology Author(s): Y. Qiu, S. Myong 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.