title: A lifetime-sensitive fluorescence anisotropy probe for DNA-based bioassays: The case of SYBR Green.
abstract: In standard steady-state fluorescence anisotropy (FA) DNA-based assays, the ligand binding to a given receptor is typically signalled by the rotational correlation time changes of the tracer. Herein, we report a radically different strategy that relies on the peculiar excited state lifetime features of the SYBR Green (SG) dye. This DNA-binding probe exhibits a drastically short lifetime in solution, leading to a high FA signal. Its complexation to oligonucleotides determines a singular and very large depolarization depending on the concerted effects of extreme lifetime enhancement and resonance energy homotransfer. On the basis of ligand-induced changes in the molar fractions of bound and free forms of SG, the approach provides an unprecedented means for the FA monitoring of the ligand binding to short DNA molecules, allowing the elaboration of a variety of intercalator displacement assays and label-free biosensors that involve diverse DNA structures (duplex, hairpin, G-quadruplex and single-stranded), ligand types (ion, small organic molecule and protein) and binding modes (intercalation, minor groove, allosteric switch). These findings open up promising avenues in the design of a new generation of FA assays.
authors.affiliation: University Grenoble Alpes, DPM UMR 5063, F-38041 Grenoble, France; CNRS, DPM UMR 5063, F-38041 Grenoble, France; Département de Biochimie, Toxicologie et Pharmacologie, CHU de Grenoble site Nord - Institut de biologie et de pathologie, F-38041 Grenoble, France.
authors.forename: Benoit
authors.initials: B
authors.lastname: Chovelon
authors.affiliation: University Grenoble Alpes, DPM UMR 5063, F-38041 Grenoble, France; CNRS, DPM UMR 5063, F-38041 Grenoble, France.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Fiore
authors.affiliation: Département de Biochimie, Toxicologie et Pharmacologie, CHU de Grenoble site Nord - Institut de biologie et de pathologie, F-38041 Grenoble, France; University Grenoble Alpes, Laboratory of Hypoxy Physiopathology Study Inserm U1042, 38700 La Tronche, France.
authors.forename: Patrice
authors.initials: P
authors.lastname: Faure
authors.affiliation: University Grenoble Alpes, DPM UMR 5063, F-38041 Grenoble, France; CNRS, DPM UMR 5063, F-38041 Grenoble, France. Electronic address: eric.peyrin@ujf-grenoble.fr.
authors.forename: Eric
authors.initials: E
authors.lastname: Peyrin
authors.affiliation: University Grenoble Alpes, DPM UMR 5063, F-38041 Grenoble, France; CNRS, DPM UMR 5063, F-38041 Grenoble, France. Electronic address: corinne.ravelet@ujf-grenoble.fr.
authors.forename: Corinne
authors.initials: C
authors.lastname: Ravelet
mesh_terms: D052160:Benzothiazoles; D001681:Biological Assay; D015374:Biosensing Techniques / Q000379:methods*; D004247:DNA / Q000737:chemistry*; D003959:Diamines; D005454:Fluorescence Polarization / Q000379:methods*; D054856:G-Quadruplexes; D006801:Humans; D007364:Intercalating Agents / Q000737:chemistry; D008024:Ligands; D009841:Oligonucleotides / Q000737:chemistry; D009930:Organic Chemicals / Q000737:chemistry; D011804:Quinolines
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D007364:Intercalating Agents; D008024:Ligands; D009841:Oligonucleotides; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D004247:DNA
keywords: DNA oligonucleotide; Fluorescence anisotropy; Lifetime; SYBR Green; Wide applicability