Single-molecule Förster resonance energy transfer (smFRET) allows measuring distances between donor and acceptor fluorophores on the 3-10 nm range. Solution-based smFRET allows measurement of binding-unbinding events or conformational changes of dye-labeled biomolecules without ensemble averaging and free from surface perturbations. When employing dual (or multi) laser excitation, smFRET allows resolving the number of fluorescent labels on each molecule, greatly enhancing the ability to study heterogeneous samples. A major drawback to solution-based smFRET is the low throughput, which renders repetitive measurements expensive and hinders the ability to study kinetic phenomena in real-time. Here we demonstrate a high-throughput smFRET system that multiplexes acquisition by using 48 excitation spots and two 48-pixel single-photon avalanche diode array detectors. The system employs two excitation lasers allowing separation of species with one or two active fluorophores. The performance of the system is demonstrated on a set of doubly labeled double-stranded DNA oligonucleotides with different distances between donor and acceptor dyes along the DNA duplex. We show that the acquisition time for accurate subpopulation identification is reduced from several minutes to seconds, opening the way to high-throughput screening applications and real-time kinetics studies of enzymatic reactions such as DNA transcription by bacterial RNA polymerase.
|Journal||Journal of Chemical Physics|
|State||Published - 28 Mar 2018|
Bibliographical noteFunding Information:
The authors thank Luca Miari for help in the initial stage of this project, Mr. Yazan Alhadid and Dr. Eitan Lerner for help with single-molecule sample preparation, and Dr. Eitan Lerner for critical reading of the manuscript. We thank Dr. Bentolila for the generous loan of a LCOS-SLM from the Advanced Light Microscopy/Spectroscopy Shared Resource Facility at the California NanoSystems Institute at UCLA. Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Nos. R01 GM095904 and R01 GM069709 and by the National Science Foundation under Award No. MCB 1244175. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the National Science Foundation.
© 2018 Author(s).