Rxivist logo

Multispot single-molecule FRET: high-throughput analysis of freely diffusing molecules

By Antonino Ingargiola, Eitan Lerner, SangYoon Chung, F. Panzeri, A. Gulinatti, I. Rech, M. Ghioni, Shimon Weiss, Xavier Michalet

Posted 02 Nov 2016
bioRxiv DOI: 10.1101/085027 (published DOI: 10.1371/journal.pone.0175766)

We describe an 8-spot confocal setup for high-throughput smFRET assays and illustrate its performance with two characteristic experiments. First, measurements on a series of freely diffusing doubly-labeled dsDNA samples allow us to demonstrate that data acquired in multiple spots in parallel can be properly corrected and result in measured sample characteristics identical to those obtained with a standard single-spot setup. We then take advantage of the higher throughput provided by parallel acquisition to address an outstanding question about the kinetics of the initial steps of bacterial RNA transcription. Our real-time kinetic analysis of promoter escape by bacterial RNA polymerase confirms results obtained by a more indirect route, shedding additional light on the initial steps of transcription. Finally, we discuss the advantages of our multispot setup, while pointing potential limitations of the current single laser excitation design, as well as analysis challenges and their solutions.

Download data

  • Downloaded 729 times
  • Download rankings, all-time:
    • Site-wide: 20,030 out of 94,912
    • In biophysics: 690 out of 4,144
  • Year to date:
    • Site-wide: 48,611 out of 94,912
  • Since beginning of last month:
    • Site-wide: 33,422 out of 94,912

Altmetric data


Downloads over time

Distribution of downloads per paper, site-wide


PanLingua

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


News