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

Buscaglia R, Jameson DM, Chaires JB - Nucleic Acids Res. (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 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.

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Construction of a phasor point for a mixture of two single-exponential lifetimes in solution. Phase and modulation data were simulated using a frequency of 70 MHz. The phasor points corresponding to single exponential decays of 10 ns (closed circle) and 1 ns (open circle) fall on the universal circle. A mixture of these two fluorophores in solution would result in two exponential decays, resulting in a phasor point falling within the universal circle. The phasor point would fall along the line segment connecting the two unique single exponential decays, with the position of the point dependent on the fractional intensity composition of the mixture. The phasor point shown (closed box) represents 1:1 fractional intensities of 10 and 1 ns species in solution, resulting in a phasor point that lies directly between the two points on the universal circle.
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gkr1286-F1: Construction of a phasor point for a mixture of two single-exponential lifetimes in solution. Phase and modulation data were simulated using a frequency of 70 MHz. The phasor points corresponding to single exponential decays of 10 ns (closed circle) and 1 ns (open circle) fall on the universal circle. A mixture of these two fluorophores in solution would result in two exponential decays, resulting in a phasor point falling within the universal circle. The phasor point would fall along the line segment connecting the two unique single exponential decays, with the position of the point dependent on the fractional intensity composition of the mixture. The phasor point shown (closed box) represents 1:1 fractional intensities of 10 and 1 ns species in solution, resulting in a phasor point that lies directly between the two points on the universal circle.

Mentions: The placement of the phasor point of a complex mixture can be described as the linear combination of single exponential phasor points (Figure 1). As stated earlier, two fluorophores having single exponential lifetimes will exhibit phasor points that fall along the universal circle. A mixture of the single-exponential fluorophores will result in a phasor point that lies at a position within the universal circle along the line segment connecting the two single exponential phasor points (16). Importantly, any movement in the position of the phasor point indicates changes in the lifetimes or fractional intensities. Sensitive time-dependent measurements are simplified to movements of the phasor point that establish interactions and dynamics of the system. Phasors allow for rapid interpretation of time-dependent lifetime results and can be used to determine changes in the local environment of the fluorophore being monitored, resulting in simplified analysis of fluorophore mixtures, structural changes and binding interactions.Figure 1.


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

Buscaglia R, Jameson DM, Chaires JB - Nucleic Acids Res. (2012)

Construction of a phasor point for a mixture of two single-exponential lifetimes in solution. Phase and modulation data were simulated using a frequency of 70 MHz. The phasor points corresponding to single exponential decays of 10 ns (closed circle) and 1 ns (open circle) fall on the universal circle. A mixture of these two fluorophores in solution would result in two exponential decays, resulting in a phasor point falling within the universal circle. The phasor point would fall along the line segment connecting the two unique single exponential decays, with the position of the point dependent on the fractional intensity composition of the mixture. The phasor point shown (closed box) represents 1:1 fractional intensities of 10 and 1 ns species in solution, resulting in a phasor point that lies directly between the two points on the universal circle.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351194&req=5

gkr1286-F1: Construction of a phasor point for a mixture of two single-exponential lifetimes in solution. Phase and modulation data were simulated using a frequency of 70 MHz. The phasor points corresponding to single exponential decays of 10 ns (closed circle) and 1 ns (open circle) fall on the universal circle. A mixture of these two fluorophores in solution would result in two exponential decays, resulting in a phasor point falling within the universal circle. The phasor point would fall along the line segment connecting the two unique single exponential decays, with the position of the point dependent on the fractional intensity composition of the mixture. The phasor point shown (closed box) represents 1:1 fractional intensities of 10 and 1 ns species in solution, resulting in a phasor point that lies directly between the two points on the universal circle.
Mentions: The placement of the phasor point of a complex mixture can be described as the linear combination of single exponential phasor points (Figure 1). As stated earlier, two fluorophores having single exponential lifetimes will exhibit phasor points that fall along the universal circle. A mixture of the single-exponential fluorophores will result in a phasor point that lies at a position within the universal circle along the line segment connecting the two single exponential phasor points (16). Importantly, any movement in the position of the phasor point indicates changes in the lifetimes or fractional intensities. Sensitive time-dependent measurements are simplified to movements of the phasor point that establish interactions and dynamics of the system. Phasors allow for rapid interpretation of time-dependent lifetime results and can be used to determine changes in the local environment of the fluorophore being monitored, resulting in simplified analysis of fluorophore mixtures, structural changes and binding interactions.Figure 1.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 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.

Show MeSH
Related in: MedlinePlus