<i>iMAX</i> FRET (Information Maximized FRET) for Multipoint Single-Molecule Structural Analysis.

Abstract

Understanding the structure of biomolecules is vital for deciphering their roles in biological systems. Single-molecule techniques have emerged as alternatives to conventional ensemble structure analysis methods for uncovering new biology in molecular dynamics and interaction studies, yet only limited structural information could be obtained experimentally. Here, we address this challenge by introducing <i>iMAX</i> FRET, a one-pot method that allows <i>ab initio</i> 3D profiling of individual molecules using two-color FRET measurements. Through the stochastic exchange of fluorescent weak binders, <i>iMAX</i> FRET simultaneously assesses multiple distances on a biomolecule within a few minutes, which can then be used to reconstruct the coordinates of up to four points in each molecule, allowing structure-based inference. We demonstrate the 3D reconstruction of DNA nanostructures, protein quaternary structures, and conformational changes in proteins. With <i>iMAX</i> FRET, we provide a powerful approach to advance the understanding of biomolecular structure by expanding conventional FRET analysis to three dimensions.