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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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G-quadruplex folding monitored through changes in FRET efficiencies using donor alone (FAM) and donor-acceptor (FAM-TAMRA) labeled human telomeric 22-base sequences upon the addition of potassium (circles) or sodium (boxes). (A) G-quadruplex folding was monitored by total fluorescence intensity upon the addition of cation over a range of 0–100 mM. Both potassium and sodium show a single significant transition with midpoints at 5 and 25 mM, respectively. The background intensities from potassium (filled circles) and sodium (filled boxes) titrations using donor alone show minimal changes in total intensity. (B) Phasor diagrams using 93 MHz frequency were prepared from the same titrations used for total intensity. Solid arching line represents the universal circle. The donor alone samples (closed circles and squares) show no movement and remain near the universal circle with increasing cation concentration, whereas dual-labeled telomeric 22-base DNA shows phasor points distinct from donor alone and significant trajectories with increasing cation concentration. (C) Enlarged phasor diagrams emphasizing motion of the phasor point with cation titrations. Potassium phasors (circles) show two unique trajectories with movement downward over the potassium range 3–30 mM and movement right from 30 to 100 mM. Sodium (squares) shows only a single trajectory with significant movement over the range 15–100 mM.
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gkr1286-F7: G-quadruplex folding monitored through changes in FRET efficiencies using donor alone (FAM) and donor-acceptor (FAM-TAMRA) labeled human telomeric 22-base sequences upon the addition of potassium (circles) or sodium (boxes). (A) G-quadruplex folding was monitored by total fluorescence intensity upon the addition of cation over a range of 0–100 mM. Both potassium and sodium show a single significant transition with midpoints at 5 and 25 mM, respectively. The background intensities from potassium (filled circles) and sodium (filled boxes) titrations using donor alone show minimal changes in total intensity. (B) Phasor diagrams using 93 MHz frequency were prepared from the same titrations used for total intensity. Solid arching line represents the universal circle. The donor alone samples (closed circles and squares) show no movement and remain near the universal circle with increasing cation concentration, whereas dual-labeled telomeric 22-base DNA shows phasor points distinct from donor alone and significant trajectories with increasing cation concentration. (C) Enlarged phasor diagrams emphasizing motion of the phasor point with cation titrations. Potassium phasors (circles) show two unique trajectories with movement downward over the potassium range 3–30 mM and movement right from 30 to 100 mM. Sodium (squares) shows only a single trajectory with significant movement over the range 15–100 mM.

Mentions: Potassium and sodium titrations were completed using a FAM/TAMRA dual-labeled 22-nt human telomeric G-quadruplex forming sequence where both the emission intensity and frequency-domain lifetime measurements were collected (Figure 7). Filter sets were used for the collection of fluorescence emission of only FAM. Total intensity measurements using the FAM/TAMRA ODN show a decrease in the emission intensity of FAM upon the introduction of cation. This decrease in fluorescence emission intensity is caused by the formation of G-quadruplex, bringing the FAM/TAMRA pair closer together and allowing for increased efficiency of energy transfer. This change is distinct from titrations conducted using donor-alone ODN. When the 22-nt ODN is labeled with only FAM, the change in total intensity is <10% (Figure 7A). This small change with the FAM-only ODN indicates the change seen from the FAM/TAMRA dual-labeled ODN corresponds to energy transfer and not an influence on the fluorescence intensity due to the addition of cation. The potassium titration has a midpoint of 5 mM, similar to that found from the 2AP titrations, whereas the sodium titration has a significantly higher midpoint at 25 mM. These results match previous telomeric G-quadruplex folding studies (41).Figure 7.


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

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

G-quadruplex folding monitored through changes in FRET efficiencies using donor alone (FAM) and donor-acceptor (FAM-TAMRA) labeled human telomeric 22-base sequences upon the addition of potassium (circles) or sodium (boxes). (A) G-quadruplex folding was monitored by total fluorescence intensity upon the addition of cation over a range of 0–100 mM. Both potassium and sodium show a single significant transition with midpoints at 5 and 25 mM, respectively. The background intensities from potassium (filled circles) and sodium (filled boxes) titrations using donor alone show minimal changes in total intensity. (B) Phasor diagrams using 93 MHz frequency were prepared from the same titrations used for total intensity. Solid arching line represents the universal circle. The donor alone samples (closed circles and squares) show no movement and remain near the universal circle with increasing cation concentration, whereas dual-labeled telomeric 22-base DNA shows phasor points distinct from donor alone and significant trajectories with increasing cation concentration. (C) Enlarged phasor diagrams emphasizing motion of the phasor point with cation titrations. Potassium phasors (circles) show two unique trajectories with movement downward over the potassium range 3–30 mM and movement right from 30 to 100 mM. Sodium (squares) shows only a single trajectory with significant movement over the range 15–100 mM.
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gkr1286-F7: G-quadruplex folding monitored through changes in FRET efficiencies using donor alone (FAM) and donor-acceptor (FAM-TAMRA) labeled human telomeric 22-base sequences upon the addition of potassium (circles) or sodium (boxes). (A) G-quadruplex folding was monitored by total fluorescence intensity upon the addition of cation over a range of 0–100 mM. Both potassium and sodium show a single significant transition with midpoints at 5 and 25 mM, respectively. The background intensities from potassium (filled circles) and sodium (filled boxes) titrations using donor alone show minimal changes in total intensity. (B) Phasor diagrams using 93 MHz frequency were prepared from the same titrations used for total intensity. Solid arching line represents the universal circle. The donor alone samples (closed circles and squares) show no movement and remain near the universal circle with increasing cation concentration, whereas dual-labeled telomeric 22-base DNA shows phasor points distinct from donor alone and significant trajectories with increasing cation concentration. (C) Enlarged phasor diagrams emphasizing motion of the phasor point with cation titrations. Potassium phasors (circles) show two unique trajectories with movement downward over the potassium range 3–30 mM and movement right from 30 to 100 mM. Sodium (squares) shows only a single trajectory with significant movement over the range 15–100 mM.
Mentions: Potassium and sodium titrations were completed using a FAM/TAMRA dual-labeled 22-nt human telomeric G-quadruplex forming sequence where both the emission intensity and frequency-domain lifetime measurements were collected (Figure 7). Filter sets were used for the collection of fluorescence emission of only FAM. Total intensity measurements using the FAM/TAMRA ODN show a decrease in the emission intensity of FAM upon the introduction of cation. This decrease in fluorescence emission intensity is caused by the formation of G-quadruplex, bringing the FAM/TAMRA pair closer together and allowing for increased efficiency of energy transfer. This change is distinct from titrations conducted using donor-alone ODN. When the 22-nt ODN is labeled with only FAM, the change in total intensity is <10% (Figure 7A). This small change with the FAM-only ODN indicates the change seen from the FAM/TAMRA dual-labeled ODN corresponds to energy transfer and not an influence on the fluorescence intensity due to the addition of cation. The potassium titration has a midpoint of 5 mM, similar to that found from the 2AP titrations, whereas the sodium titration has a significantly higher midpoint at 25 mM. These results match previous telomeric G-quadruplex folding studies (41).Figure 7.

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