Single-molecule fluorescence resonance energy transfer (smFRET) has proven to be a uniquely powerful tool to study the structural dynamics of biomolecular systems which underpin their function. Until relatively recently, smFRET studies have been hampered by low-throughput and comparative studies have required consecutive measurements of individual samples. Consequently, experimental variances can be introduced that may obscure functional distinctions in systems with subtly different kinetic behavior. Here, we introduce parallel rapid exchange (PRE) smFRET, which enables head-to-head comparative investigations of a range of biomolecular systems under identical conditions at both steady state and pre-steady state. This approach combines microarray printing with DNA-barcoded immobilization to site-specifically pull-down molecules of interest from a complex mixture, such that multiple distinct samples can be imaged within one FOV. Computer-controlled fluidics enables rapid exchange of solution during imaging, increasing reproducibility and throughput for the study of pre-equilibrium processes. Additionally, PRE-smFRET is broadly applicable to a range of biomolecular systems including nucleic acids, proteins, and RNA-protein complexes. We utilize PRE-smFRET to (1) study the order and timing of activating conformational changes in a key terminator of GPCR-mediated signaling, (2) uncover the kinetic mechanism of antibiotic induced misreading of mRNA by the bacterial ribosome, and (3) demonstrate that ribosomes carrying endogenously encoded rRNA sequence variation are differentially sensitive to tetracycline class antibiotics in vitro—recapitulating in vivo studies. PRE-smFRET increases experimental throughput, reduces experimental variances, and offers an approach that can illuminate subtle kinetic distinctions across a broad range of diverse biological systems.
Brady et al. (Sun,) studied this question.