title: G-quadruplex structure and stability illuminated by 2-aminopurine phasor plots.
abstract: The use of time-resolved fluorescence measurements in studies of telomeric G-quadruplex folding and stability has been hampered by the complexity of fluorescence lifetime distributions in solution. The application of phasor diagrams to the analysis of time-resolved fluorescence measurements, collected from either frequency-domain or time-domain instrumentation, allows for rapid characterization of complex lifetime distributions. Phasor diagrams are model-free graphical representations of transformed time-resolved fluorescence results. Simplification of complex fluorescent decays by phasor diagrams is demonstrated here using a 2-aminopurine substituted telomeric G-quadruplex sequence. The application of phasor diagrams to complex systems is discussed with comparisons to traditional non-linear regression model fitting. Phasor diagrams allow for the folding and stability of the telomeric G-quadruplex to be monitored in the presence of either sodium or potassium. Fluorescence lifetime measurements revealed multiple transitions upon folding of the telomeric G-quadruplex through the addition of potassium. Enzymatic digestion of the telomeric G-quadruplex structure, fluorescence quenching and Förster resonance energy transfer were also monitored through phasor diagrams. This work demonstrates the sensitivity of time-resolved methods for monitoring changes to the telomeric G-quadruplex and outlines the phasor diagram approach for analysis of complex time-resolved results that can be extended to other G-quadruplex and nucleic acid systems.
authors.affiliation: James Graham Brown Cancer Center, University of Louisville, 505 S. Hancock Street, Louisville, KY 40202, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Buscaglia
authors.forename: David M
authors.initials: DM
authors.lastname: Jameson
authors.forename: Jonathan B
authors.initials: JB
authors.lastname: Chaires
mesh_terms: D015075:2-Aminopurine / Q000737:chemistry*; D004247:DNA / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D054856:G-Quadruplexes; D010940:Plant Proteins / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D016615:Telomere / Q000737:chemistry*
chemical_list: C526101:Mung Bean Nuclease; D010940:Plant Proteins; D015075:2-Aminopurine; D004247:DNA; D015719:Single-Strand Specific DNA and RNA Endonucleases