High-throughput smFRET analysis of freely diffusing nucleic acid molecules and associated proteins
Jonathan A. White,
Aaron M. Streets,
Posted 27 May 2019
bioRxiv DOI: 10.1101/651869 (published DOI: 10.1016/j.ymeth.2019.07.021)
Posted 27 May 2019
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.
- Downloaded 688 times
- Download rankings, all-time:
- Site-wide: 21,830 out of 94,912
- In biophysics: 785 out of 4,144
- Year to date:
- Site-wide: 17,433 out of 94,912
- Since beginning of last month:
- Site-wide: 18,195 out of 94,912
Downloads over time
Distribution of downloads per paper, site-wide
- 18 Dec 2019: We're pleased to announce PanLingua, a new tool that enables you to search for machine-translated bioRxiv preprints using more than 100 different languages.
- 21 May 2019: PLOS Biology has published a community page about Rxivist.org and its design.
- 10 May 2019: The paper analyzing the Rxivist dataset has been published at eLife.
- 1 Mar 2019: We now have summary statistics about bioRxiv downloads and submissions.
- 8 Feb 2019: Data from Altmetric is now available on the Rxivist details page for every preprint. Look for the "donut" under the download metrics.
- 30 Jan 2019: preLights has featured the Rxivist preprint and written about our findings.
- 22 Jan 2019: Nature just published an article about Rxivist and our data.
- 13 Jan 2019: The Rxivist preprint is live!