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48-spot single-molecule FRET setup with periodic acceptor excitation.
Ingargiola, Antonino; Segal, Maya; Gulinatti, Angelo; Rech, Ivan; Labanca, Ivan; Maccagnani, Piera; Ghioni, Massimo; Weiss, Shimon; Michalet, Xavier.
Afiliación
  • Ingargiola A; Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, USA.
  • Segal M; Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, USA.
  • Gulinatti A; Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano, Milan, Italy.
  • Rech I; Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano, Milan, Italy.
  • Labanca I; Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano, Milan, Italy.
  • Maccagnani P; Istituto per la Microelettronica e Microsistemi, IMM-CNR, Bologna, Italy.
  • Ghioni M; Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano, Milan, Italy.
  • Weiss S; Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, USA.
  • Michalet X; Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, USA.
J Chem Phys ; 148(12): 123304, 2018 Mar 28.
Article en En | MEDLINE | ID: mdl-29604810
Single-molecule Förster resonance energy transfer (smFRET) allows measuring distances between donor and acceptor 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 that multiplexes acquisition by using 48 excitation spots and two 48-pixel single-photon avalanche diode 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 donor and acceptor 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.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: ADN / Transferencia Resonante de Energía de Fluorescencia / Colorantes Fluorescentes Tipo de estudio: Prognostic_studies Idioma: En Revista: J Chem Phys Año: 2018 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: ADN / Transferencia Resonante de Energía de Fluorescencia / Colorantes Fluorescentes Tipo de estudio: Prognostic_studies Idioma: En Revista: J Chem Phys Año: 2018 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos