RT Journal Article
SR Electronic
T1 High-throughput smFRET analysis of freely diffusing nucleic acid molecules and associated proteins
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 651869
DO 10.1101/651869
A1 Maya Segal
A1 Antonino Ingargiola
A1 Eitan Lerner
A1 Sang Yoon Chung
A1 Jonathan A. White
A1 Aaron Streets
A1 S. Weiss
A1 X. Michalet
YR 2019
UL http://biorxiv.org/content/early/2019/06/25/651869.abstract
AB Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique for nanometer-scale studies of single molecules. Solution-based smFRET, in particular, can be used to study equilibrium intra- and intermolecular conformations, binding/unbinding events and conformational changes under biologically relevant conditions without ensemble averaging. However, single-spot smFRET measurements in solution are slow. Here, we detail a high-throughput smFRET approach that extends the traditional single-spot confocal geometry to a multispot one. The excitation spots are optically conjugated to two custom silicon single photon avalanche diode (SPAD) arrays. Two-color excitation is implemented using a periodic acceptor excitation (PAX), allowing distinguishing between singly- and doubly-labeled molecules. We demonstrate the ability of this setup to rapidly and accurately determine FRET efficiencies and population stoichiometries by pooling the data collected independently from the multiple spots. We also show how the high throughput of this approach can be used to increase the temporal resolution of single-molecule FRET population characterization from minutes to seconds. Combined with microfluidics, this high-throughput approach will enable simple real-time kinetic studies as well as powerful molecular screening applications.