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Nucleic Acids Res. 2012 May;40(9):4203-15. doi: 10.1093/nar/gkr1286. Epub 2012 Jan 12.

G-quadruplex structure and stability illuminated by 2-aminopurine phasor plots.

Author information

1
James Graham Brown Cancer Center, University of Louisville, 505 S. Hancock Street, Louisville, KY 40202, USA.

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.

PMID:
22241767
PMCID:
PMC3351194
DOI:
10.1093/nar/gkr1286
[Indexed for MEDLINE]
Free PMC Article

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