Single-Molecule Kinetic Studies of Nucleic Acids by Förster Resonance Energy Transfer

Abstract

Single-molecule microscopy is often used to observe and characterize the conformational dynamics of nucleic acids (NA). Due to the large variety of NA structures and the challenges specific to single-molecule observation techniques, the data recorded in such experiments must be processed via multiple statistical treatments to finally yield a reliable mechanistic view of the NA dynamics. In this chapter, we propose a comprehensive protocol to analyze single-molecule trajectories in the scope of single-molecule Förster resonance energy transfer (FRET) microscopy. The suggested protocol yields the conformational states common to all molecules in the investigated sample, together with the associated conformational transition kinetics. The given model resolves states that are indistinguishable by their observed FRET signals and is estimated with 95% confidence using error calculations on FRET states and transition rate constants. In the end, a step-by-step user guide is given to reproduce the protocol with the Multifunctional Analysis Software to Handle single-molecule FRET data (MASH-FRET).