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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) 2AP substituted G-quadruplex forming sequences derived from the human telomeric repeat. The diagram gives the abbreviations used for the different 2AP-substituted 22-nt deoxyoligonucleotide sequences along with a color-coded format used for all phasor plots. The bold X in each sequence corresponds to the placement of the 2AP in the 22-base sequence. (B) A comparison of the chemical structures of adenine and its fluorescent analog 2AP. (C) A comparison of two published human telomeric G-quadruplex conformations (143D and 2HY9) highlighting where the 2AP-substitutions (colored spheres) would be located within these distinct G-quadruplex conformations. Blue planar rectangles represent guanine residues in the formation of G-tetrad cores. The sequentially labeled telomeric forming G-quadruplex sequences will cause the fluorescent 2AP probe to localize differently dependent on the conformation and can be used to locally probe the loop environment.
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gkr1286-F8: (A) 2AP substituted G-quadruplex forming sequences derived from the human telomeric repeat. The diagram gives the abbreviations used for the different 2AP-substituted 22-nt deoxyoligonucleotide sequences along with a color-coded format used for all phasor plots. The bold X in each sequence corresponds to the placement of the 2AP in the 22-base sequence. (B) A comparison of the chemical structures of adenine and its fluorescent analog 2AP. (C) A comparison of two published human telomeric G-quadruplex conformations (143D and 2HY9) highlighting where the 2AP-substitutions (colored spheres) would be located within these distinct G-quadruplex conformations. Blue planar rectangles represent guanine residues in the formation of G-tetrad cores. The sequentially labeled telomeric forming G-quadruplex sequences will cause the fluorescent 2AP probe to localize differently dependent on the conformation and can be used to locally probe the loop environment.

Mentions: Unlabeled and 2AP-labeled 5′-d(AGGG(TTAGGG)3) were purchased from Integrated DNA Technologies (Coralville, IA, USA). All ODNs were purchased on a 5 -µmol scale with standard desalting. Four unique 2AP-labeled ODNs were purchased with substitutions at positions 1, 7, 13 and 19 corresponding to a single adenine change to 2AP (Diagram 1). Fluorescently tagged 6FAM-d(AGGG(TTAGGG)3)-TAMRA ODNs were purchased from Sigma (St Louis, MO, USA) on a 10 -µmol scale with standard desalting and HPLC purification. ODNs are quality checked by commercial sources through the use of either MALDI-TOF or Electrospray Ionization mass spectrometry. All purchased ODNs were used without further purification. Fluorescent tag abbreviations are 6-carboxyfluorescein (6FAM) and tetramethylrhodamine (TAMRA). Prior to use, DNAs were diluted into folding buffer (10 mM tetrabutylammonium phosphate, 1 mM EDTA acid, pH 7.1) to a stock concentration of 1 mM and stored at 4°C. Folding buffer represents a potassium and sodium-free solvent resulting in minimal G-quadruplex formation prior to the addition of added cation. Folding buffer solutions are used to provide a defined initial state so the effects of added cation can be clearly identified. Unless otherwise stated, all experimental samples were annealed prior to use by placement in boiling water for 10 min followed by quenching on ice for 10 min. All fluorescent standards and reagents were purchased at the highest available grade from Sigma. Fluorescent standards were prepared at stock concentrations of 1 mg/ml and diluted to experimental concentrations for matched intensities with samples using the following solvents: p-terphenyl (τ = 1.05 ns) in ethanol, fluorescein (τ = 4.1 ns) in 0.1 M NaOH and 2AP (τ = 10.5 ns) in folding buffer. Stock solutions of 1 M NaCl, 1 M KCl and 3 M acrylamide were prepared in folding buffer.Diagram 1.


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

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

(A) 2AP substituted G-quadruplex forming sequences derived from the human telomeric repeat. The diagram gives the abbreviations used for the different 2AP-substituted 22-nt deoxyoligonucleotide sequences along with a color-coded format used for all phasor plots. The bold X in each sequence corresponds to the placement of the 2AP in the 22-base sequence. (B) A comparison of the chemical structures of adenine and its fluorescent analog 2AP. (C) A comparison of two published human telomeric G-quadruplex conformations (143D and 2HY9) highlighting where the 2AP-substitutions (colored spheres) would be located within these distinct G-quadruplex conformations. Blue planar rectangles represent guanine residues in the formation of G-tetrad cores. The sequentially labeled telomeric forming G-quadruplex sequences will cause the fluorescent 2AP probe to localize differently dependent on the conformation and can be used to locally probe the loop environment.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1286-F8: (A) 2AP substituted G-quadruplex forming sequences derived from the human telomeric repeat. The diagram gives the abbreviations used for the different 2AP-substituted 22-nt deoxyoligonucleotide sequences along with a color-coded format used for all phasor plots. The bold X in each sequence corresponds to the placement of the 2AP in the 22-base sequence. (B) A comparison of the chemical structures of adenine and its fluorescent analog 2AP. (C) A comparison of two published human telomeric G-quadruplex conformations (143D and 2HY9) highlighting where the 2AP-substitutions (colored spheres) would be located within these distinct G-quadruplex conformations. Blue planar rectangles represent guanine residues in the formation of G-tetrad cores. The sequentially labeled telomeric forming G-quadruplex sequences will cause the fluorescent 2AP probe to localize differently dependent on the conformation and can be used to locally probe the loop environment.
Mentions: Unlabeled and 2AP-labeled 5′-d(AGGG(TTAGGG)3) were purchased from Integrated DNA Technologies (Coralville, IA, USA). All ODNs were purchased on a 5 -µmol scale with standard desalting. Four unique 2AP-labeled ODNs were purchased with substitutions at positions 1, 7, 13 and 19 corresponding to a single adenine change to 2AP (Diagram 1). Fluorescently tagged 6FAM-d(AGGG(TTAGGG)3)-TAMRA ODNs were purchased from Sigma (St Louis, MO, USA) on a 10 -µmol scale with standard desalting and HPLC purification. ODNs are quality checked by commercial sources through the use of either MALDI-TOF or Electrospray Ionization mass spectrometry. All purchased ODNs were used without further purification. Fluorescent tag abbreviations are 6-carboxyfluorescein (6FAM) and tetramethylrhodamine (TAMRA). Prior to use, DNAs were diluted into folding buffer (10 mM tetrabutylammonium phosphate, 1 mM EDTA acid, pH 7.1) to a stock concentration of 1 mM and stored at 4°C. Folding buffer represents a potassium and sodium-free solvent resulting in minimal G-quadruplex formation prior to the addition of added cation. Folding buffer solutions are used to provide a defined initial state so the effects of added cation can be clearly identified. Unless otherwise stated, all experimental samples were annealed prior to use by placement in boiling water for 10 min followed by quenching on ice for 10 min. All fluorescent standards and reagents were purchased at the highest available grade from Sigma. Fluorescent standards were prepared at stock concentrations of 1 mg/ml and diluted to experimental concentrations for matched intensities with samples using the following solvents: p-terphenyl (τ = 1.05 ns) in ethanol, fluorescein (τ = 4.1 ns) in 0.1 M NaOH and 2AP (τ = 10.5 ns) in folding buffer. Stock solutions of 1 M NaCl, 1 M KCl and 3 M acrylamide were prepared in folding buffer.Diagram 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