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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) Stern–Volmer quenching plots constructed from fluorescent intensity (F0/F—filled squares) and average lifetime (τ0/τ—open circles) for the addition of acrylamide to 13AP. The Stern–Volmer plots show downward curvature for both intensity and average lifetime indicating multiple accessible 2AP populations. (B) Stern–Volmer plots comparing acrylamide quenching of free 2AP (triangles) with the fluorescent intensity and averaged lifetime of 13AP. The quenching of free 2AP shows diffusion limited accessibility to quenching by acrylamide. (C) Quenching of 1AP (filled circles) and 13AP (open boxes) by acrylamide monitored using phasors at 49 MHz. Arrows indicate the direction of increasing acrylamide concentration. Phasor diagram shows an initial fast change in phasor position followed by a slower moving phasor change. The quenching of free 2AP (stars) by acrylamide analyzed by phasor diagrams is located initially on the universal circle with slow deviation from the universal circle at high acrylamide concentrations. This is proposed to be a similar deviation from a single lifetime component as was previously reported in glycerol/water mixtures (10), or small background interference being seen at high quencher concentrations.
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gkr1286-F6: (A) Stern–Volmer quenching plots constructed from fluorescent intensity (F0/F—filled squares) and average lifetime (τ0/τ—open circles) for the addition of acrylamide to 13AP. The Stern–Volmer plots show downward curvature for both intensity and average lifetime indicating multiple accessible 2AP populations. (B) Stern–Volmer plots comparing acrylamide quenching of free 2AP (triangles) with the fluorescent intensity and averaged lifetime of 13AP. The quenching of free 2AP shows diffusion limited accessibility to quenching by acrylamide. (C) Quenching of 1AP (filled circles) and 13AP (open boxes) by acrylamide monitored using phasors at 49 MHz. Arrows indicate the direction of increasing acrylamide concentration. Phasor diagram shows an initial fast change in phasor position followed by a slower moving phasor change. The quenching of free 2AP (stars) by acrylamide analyzed by phasor diagrams is located initially on the universal circle with slow deviation from the universal circle at high acrylamide concentrations. This is proposed to be a similar deviation from a single lifetime component as was previously reported in glycerol/water mixtures (10), or small background interference being seen at high quencher concentrations.

Mentions: Another important fluorescence technique in the study of G-quadruplex structure is collisional quenching (7). The accessibility of singly substituted 2AP residues to quenching by acrylamide has been used to probe the loops of human telomeric G-quadruplexes (43). Stern–Volmer plots constructed for acrylamide quenching of the 13AP telomeric G-quadruplex show characteristic downward curvature, distinct from the quenching of high accessible 2AP free in solution (Figure 6A). Downward curvature of the Stern–Volmer quenching plots indicates heterogeneity in the 2AP environment, which can result from either multiple global G-quadruplex conformations or distinct localized position of the fluorophore within the G-quadruplex loop. The downward curvature of these Stern–Volmer plots is distinct from the quenching of 2AP nucleotides free in solution, which demonstrate a linear dependence to added quencher and were determined to have a diffusion limited quenching constant (Figure 6B).Figure 6.


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

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

(A) Stern–Volmer quenching plots constructed from fluorescent intensity (F0/F—filled squares) and average lifetime (τ0/τ—open circles) for the addition of acrylamide to 13AP. The Stern–Volmer plots show downward curvature for both intensity and average lifetime indicating multiple accessible 2AP populations. (B) Stern–Volmer plots comparing acrylamide quenching of free 2AP (triangles) with the fluorescent intensity and averaged lifetime of 13AP. The quenching of free 2AP shows diffusion limited accessibility to quenching by acrylamide. (C) Quenching of 1AP (filled circles) and 13AP (open boxes) by acrylamide monitored using phasors at 49 MHz. Arrows indicate the direction of increasing acrylamide concentration. Phasor diagram shows an initial fast change in phasor position followed by a slower moving phasor change. The quenching of free 2AP (stars) by acrylamide analyzed by phasor diagrams is located initially on the universal circle with slow deviation from the universal circle at high acrylamide concentrations. This is proposed to be a similar deviation from a single lifetime component as was previously reported in glycerol/water mixtures (10), or small background interference being seen at high quencher concentrations.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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gkr1286-F6: (A) Stern–Volmer quenching plots constructed from fluorescent intensity (F0/F—filled squares) and average lifetime (τ0/τ—open circles) for the addition of acrylamide to 13AP. The Stern–Volmer plots show downward curvature for both intensity and average lifetime indicating multiple accessible 2AP populations. (B) Stern–Volmer plots comparing acrylamide quenching of free 2AP (triangles) with the fluorescent intensity and averaged lifetime of 13AP. The quenching of free 2AP shows diffusion limited accessibility to quenching by acrylamide. (C) Quenching of 1AP (filled circles) and 13AP (open boxes) by acrylamide monitored using phasors at 49 MHz. Arrows indicate the direction of increasing acrylamide concentration. Phasor diagram shows an initial fast change in phasor position followed by a slower moving phasor change. The quenching of free 2AP (stars) by acrylamide analyzed by phasor diagrams is located initially on the universal circle with slow deviation from the universal circle at high acrylamide concentrations. This is proposed to be a similar deviation from a single lifetime component as was previously reported in glycerol/water mixtures (10), or small background interference being seen at high quencher concentrations.
Mentions: Another important fluorescence technique in the study of G-quadruplex structure is collisional quenching (7). The accessibility of singly substituted 2AP residues to quenching by acrylamide has been used to probe the loops of human telomeric G-quadruplexes (43). Stern–Volmer plots constructed for acrylamide quenching of the 13AP telomeric G-quadruplex show characteristic downward curvature, distinct from the quenching of high accessible 2AP free in solution (Figure 6A). Downward curvature of the Stern–Volmer quenching plots indicates heterogeneity in the 2AP environment, which can result from either multiple global G-quadruplex conformations or distinct localized position of the fluorophore within the G-quadruplex loop. The downward curvature of these Stern–Volmer plots is distinct from the quenching of 2AP nucleotides free in solution, which demonstrate a linear dependence to added quencher and were determined to have a diffusion limited quenching constant (Figure 6B).Figure 6.

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