Format

Send to

Choose Destination
Methods. 2019 Jul 26. pii: S1046-2023(18)30443-2. doi: 10.1016/j.ymeth.2019.07.021. [Epub ahead of print]

High-throughput smFRET analysis of freely diffusing nucleic acid molecules and associated proteins.

Author information

1
Department of Chemistry & Biochemistry, UCLA, Los Angeles, CA 90095, USA.
2
Department of Chemistry & Biochemistry, UCLA, Los Angeles, CA 90095, USA; Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Faculty of Mathematics & Science, The Edmond J. Safra Campus, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel.
3
Department of Bioengineering, UC Berkeley, Berkeley, CA 94720, USA.
4
Department of Bioengineering, UC Berkeley, Berkeley, CA 94720, USA; Chan-Zuckerberg Biohub, San Francisco, CA 94158, USA.
5
Department of Chemistry & Biochemistry, UCLA, Los Angeles, CA 90095, USA. Electronic address: michalet@chem.ucla.edu.

Abstract

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 o 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.

KEYWORDS:

Freely-diffusing; High-throughput; SPAD array; Single-molecule FRET

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center