High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence

Zero-mode waveguides provide a powerful technology for studying single-molecule real-time dynamics of biological systems at physiological ligand concentrations. We customized a commercial zero-mode waveguide-based DNA sequencer for use as a versatile instrument for single-molecule fluorescence detec...

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Published inProceedings of the National Academy of Sciences - PNAS Vol. 111; no. 2; pp. 664 - 669
Main Authors Chen, Jin, Dalal, Ravindra V., Petrov, Alexey N., Tsai, Albert, O'Leary, Seán E., Chapin, Karen, Cheng, Janice, Ewan, Mark, Hsiung, Pei-Lin, Lundquist, Paul, Turner, Stephen W., Hsu, David R., Puglisi, Joseph D.
Format Journal Article
LanguageEnglish
Published United States National Academy of Sciences 14.01.2014
National Acad Sciences
Subjects
Algorithms
Biochemistry
Biological Sciences
Biophysics
Biophysics - instrumentation
Biophysics - methods
Codons
Computer Systems
Dyes
Fluorescence
High-Throughput Screening Assays - instrumentation
High-Throughput Screening Assays - methods
Lasers
Ligands
Messenger RNA
Molecular biology
Molecules
Monitoring, Physiologic - instrumentation
Monitoring, Physiologic - methods
Physical Sciences
Reagents
Ribosomes
Software
Transfer RNA
Waveguides
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Summary:Zero-mode waveguides provide a powerful technology for studying single-molecule real-time dynamics of biological systems at physiological ligand concentrations. We customized a commercial zero-mode waveguide-based DNA sequencer for use as a versatile instrument for single-molecule fluorescence detection and showed that the system provides long fluorophore lifetimes with good signal to noise and low spectral cross-talk. We then used a ribosomal translation assay to show real-time fluidic delivery during data acquisition, showing it is possible to follow the conformation and composition of thousands of single biomolecules simultaneously through four spectral channels. This instrument allows high-throughput multiplexed dynamics of single-molecule biological processes over long timescales. The instrumentation presented here has broad applications to single-molecule studies of biological systems and is easily accessible to the biophysical community.
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Edited by Maxime Dahan, Institut Curie, Paris, France, and accepted by the Editorial Board December 4, 2013 (received for review August 21, 2013)
1J. Chen and R.V.D. contributed equally to this work.
Author contributions: J. Chen, R.V.D., A.N.P., A.T., S.E.O., S.W.T., D.R.H., and J.D.P. designed research; J. Chen, R.V.D., K.C., J. Cheng, M.E., P.-L.H., and P.L. performed research; J. Chen, R.V.D., A.N.P., A.T., S.E.O., K.C., J. Cheng, M.E., P.-L.H., and P.L. contributed new reagents/analytic tools; J. Chen, R.V.D., K.C., J. Cheng, M.E., P.-L.H., and P.L. analyzed data; and J. Chen, R.V.D., A.N.P., A.T., S.E.O., D.R.H., and J.D.P. wrote the paper.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.1315735111