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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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(A) Phasor diagram representations at 70 MHz of the four different 2AP-labeled deoxyoligonucleotides in the presence of either potassium or sodium with colors corresponding to Diagram 1. The ‘universal circle’ is represented by a solid arching line whereas the open circle phasor point demonstrates the placement of the single exponential decay from free 2AP. (B) The evaluation of errors associated with the phasor points for both potassium and sodium highlights differences in phasor point locations dependent on the G-Quadruplex loop in which the 2AP is placed. Any difference in the phasor point reflects a 2AP environment distinct from the placement at other positions. Both potassium and sodium phasor representations show differences between the environment of all four positions used to probe the G-quadruplex conformation.
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gkr1286-F3: (A) Phasor diagram representations at 70 MHz of the four different 2AP-labeled deoxyoligonucleotides in the presence of either potassium or sodium with colors corresponding to Diagram 1. The ‘universal circle’ is represented by a solid arching line whereas the open circle phasor point demonstrates the placement of the single exponential decay from free 2AP. (B) The evaluation of errors associated with the phasor points for both potassium and sodium highlights differences in phasor point locations dependent on the G-Quadruplex loop in which the 2AP is placed. Any difference in the phasor point reflects a 2AP environment distinct from the placement at other positions. Both potassium and sodium phasor representations show differences between the environment of all four positions used to probe the G-quadruplex conformation.

Mentions: Phasor points were constructed for all four 2AP-labeled ODNs in the presence of 25 mM potassium or 50 mM sodium (Figure 3A). The four 2AP-substituted ODNs are described by points on the phasor plot corresponding to unique environments around each of the substitutions. All four 2AP-labeled ODN phasor points fall within the universal circle indicating a mixture of fluorescent lifetime decays. The phasor points are constructed through model free analysis, requiring no prior knowledge of the lifetime mixture or non-linear regression analysis. The significant advantage of these phasor plots is that they show, at a glance, a specific signature of particular G-quadruplex conformations. The signature for the Na+ ‘basket’ form is clearly distinctive from that of the K+ ‘hybrid’ form.Figure 3.


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

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

(A) Phasor diagram representations at 70 MHz of the four different 2AP-labeled deoxyoligonucleotides in the presence of either potassium or sodium with colors corresponding to Diagram 1. The ‘universal circle’ is represented by a solid arching line whereas the open circle phasor point demonstrates the placement of the single exponential decay from free 2AP. (B) The evaluation of errors associated with the phasor points for both potassium and sodium highlights differences in phasor point locations dependent on the G-Quadruplex loop in which the 2AP is placed. Any difference in the phasor point reflects a 2AP environment distinct from the placement at other positions. Both potassium and sodium phasor representations show differences between the environment of all four positions used to probe the G-quadruplex conformation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1286-F3: (A) Phasor diagram representations at 70 MHz of the four different 2AP-labeled deoxyoligonucleotides in the presence of either potassium or sodium with colors corresponding to Diagram 1. The ‘universal circle’ is represented by a solid arching line whereas the open circle phasor point demonstrates the placement of the single exponential decay from free 2AP. (B) The evaluation of errors associated with the phasor points for both potassium and sodium highlights differences in phasor point locations dependent on the G-Quadruplex loop in which the 2AP is placed. Any difference in the phasor point reflects a 2AP environment distinct from the placement at other positions. Both potassium and sodium phasor representations show differences between the environment of all four positions used to probe the G-quadruplex conformation.
Mentions: Phasor points were constructed for all four 2AP-labeled ODNs in the presence of 25 mM potassium or 50 mM sodium (Figure 3A). The four 2AP-substituted ODNs are described by points on the phasor plot corresponding to unique environments around each of the substitutions. All four 2AP-labeled ODN phasor points fall within the universal circle indicating a mixture of fluorescent lifetime decays. The phasor points are constructed through model free analysis, requiring no prior knowledge of the lifetime mixture or non-linear regression analysis. The significant advantage of these phasor plots is that they show, at a glance, a specific signature of particular G-quadruplex conformations. The signature for the Na+ ‘basket’ form is clearly distinctive from that of the K+ ‘hybrid’ form.Figure 3.

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