From Ensemble FRET to Single-Molecule Imaging: Monitoring Individual Cellular Machinery in Action

Abstract

Fluorescence resonance energy transfer is a suitable approach for observing conformational changes in biomolecules, as it is a “spectroscopic molecular ruler” sensitive to nanometer-scale distances. The experimental outcomes of FRET measurements in ensemble studies provide averaged overpopulation that could hide the details of dynamics for individual molecules. Though ensemble measurements have much application and have their advantages, including easy optical setup, easy sample preparation, etc., single-molecule methods have appeared as popular and powerful tools for the understanding of complex biophysical processes. This is because of its unique abilities to probe the molecular structure, function, and dynamics from information obtained from every individual molecule. At present, several laboratories around the world employ this technique for studying various biological systems such as cells, nucleosomes as well as important biomolecular interactions between DNA, RNA, and proteins. Single-molecule FRET assays are also widely used for studying DNA hybridization, interactions between DNA-protein, protein-protein, and protein folding, etc. The dynamic changes induced on DNA/protein substrate can easily be monitored in real-time observations.