TY - JOUR
T1 - Multispot single-molecule FRET
T2 - Highthroughput analysis of freely diffusing molecules
AU - Ingargiola, Antonino
AU - Lerner, Eitan
AU - Chung, Sangyoon
AU - Panzeri, Francesco
AU - Gulinatti, Angelo
AU - Rech, Ivan
AU - Ghioni, Massimo
AU - Weiss, Shimon
AU - Michalet, Xavier
N1 - Publisher Copyright:
© 2017 Ingargiola et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PY - 2017/4
Y1 - 2017/4
N2 - 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 consistent with 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 realtime 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.
AB - 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 consistent with 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 realtime 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.
UR - http://www.scopus.com/inward/record.url?scp=85017661655&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0175766
DO - 10.1371/journal.pone.0175766
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C2 - 28419142
AN - SCOPUS:85017661655
SN - 1932-6203
VL - 12
JO - PLoS ONE
JF - PLoS ONE
IS - 4
M1 - e0175766
ER -