Rxivist logo

Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 60,222 bioRxiv papers from 267,720 authors.

48-spot single-molecule FRET setup with periodic acceptor excitation

By Antonino Ingargiola, Maya Segal, Angelo Gulinatti, Ivan Rech, Ivan Labanca, Piera Maccagnani, Massimo Ghioni, Shimon Weiss, Xavier Michalet

Posted 26 Jun 2017
bioRxiv DOI: 10.1101/156182 (published DOI: 10.1063/1.5000742)

Single-molecule FRET (smFRET) allows measuring distances between donor (D) and acceptor (A) 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 which multiplexes acquisition by using 48 excitation spots and two 48-pixel SPAD 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 D and A 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.

Download data

  • Downloaded 573 times
  • Download rankings, all-time:
    • Site-wide: 15,481 out of 60,222
    • In biophysics: 500 out of 2,550
  • Year to date:
    • Site-wide: 42,112 out of 60,222
  • Since beginning of last month:
    • Site-wide: 58,816 out of 60,222

Altmetric data

Downloads over time

Distribution of downloads per paper, site-wide

Sign up for the Rxivist weekly newsletter! (Click here for more details.)