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Distance-dependent duplex DNA destabilization proximal to G-quadruplex/i-motif sequences.

König SL, Huppert JL, Sigel RK, Evans AC - Nucleic Acids Res. (2013)

Bottom Line: Prediction of putative G-quadruplex-forming regions is likely to be assisted by further understanding of what distance (number of base pairs) is required for duplexes to remain stable as quadruplexes or i-motifs form.Using oligonucleotide constructs derived from precedented G-quadruplexes and i-motif-forming bcl-2 P1 promoter region, initial biophysical stability studies indicate that the formation of G-quadruplex and i-motif conformations do destabilize proximal duplex regions.The undermining effect that quadruplex formation can have on duplex stability is mitigated with increased distance from the duplex region: a spacing of five base pairs or more is sufficient to maintain duplex stability proximal to predicted quadruplex/i-motif-forming regions.

View Article: PubMed Central - PubMed

Affiliation: Cavendish Laboratory, University of Cambridge, JJ Thomson Avenue, Cambridge CB3 0HE, UK, Institute of Inorganic Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland and University of Nice-Sophia Antipolis, UMR 7272 CNRS, Institut de 40 Chimie de Nice, 28 Avenue Valrose, 06108 Nice, France.

ABSTRACT
G-quadruplexes and i-motifs are complementary examples of non-canonical nucleic acid substructure conformations. G-quadruplex thermodynamic stability has been extensively studied for a variety of base sequences, but the degree of duplex destabilization that adjacent quadruplex structure formation can cause has yet to be fully addressed. Stable in vivo formation of these alternative nucleic acid structures is likely to be highly dependent on whether sufficient spacing exists between neighbouring duplex- and quadruplex-/i-motif-forming regions to accommodate quadruplexes or i-motifs without disrupting duplex stability. Prediction of putative G-quadruplex-forming regions is likely to be assisted by further understanding of what distance (number of base pairs) is required for duplexes to remain stable as quadruplexes or i-motifs form. Using oligonucleotide constructs derived from precedented G-quadruplexes and i-motif-forming bcl-2 P1 promoter region, initial biophysical stability studies indicate that the formation of G-quadruplex and i-motif conformations do destabilize proximal duplex regions. The undermining effect that quadruplex formation can have on duplex stability is mitigated with increased distance from the duplex region: a spacing of five base pairs or more is sufficient to maintain duplex stability proximal to predicted quadruplex/i-motif-forming regions.

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Thermodynamic stability of a duplex derived from the c-kit promoter in the presence and in the absence of an overhang. In the absence of a 3′ overhang (top1, bottom1) ΔG° = −67.5 kJ/mol. A 3′-poly(T) overhang decreases thermodynamic stability (ΔG° = −65.0 kJ/mol), but this decrease is not statistically significant (top1::OH1/2/3, bottom1, P > 0.05). The presence of a G-quadruplex significantly increases ΔG° (top1::GQ1/2, bottom1). Duplex destabilisation in the presence of a G-quadruplex is significant in the presence of 1 or 2 mismatches immediately proximal to the appended secondary structure motif (top1/top1::GQ2, bottom1-1MM and top1/top1::GQ2, bottom1-2MM). No significant changes are observable in the presence of three mismatches. Statistical significance of changes was estimated by analysis of variance, and Bonferroni correction for multiple testing was applied when necessary. **P < 0.01, ***P < 0.001.
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gkt476-F2: Thermodynamic stability of a duplex derived from the c-kit promoter in the presence and in the absence of an overhang. In the absence of a 3′ overhang (top1, bottom1) ΔG° = −67.5 kJ/mol. A 3′-poly(T) overhang decreases thermodynamic stability (ΔG° = −65.0 kJ/mol), but this decrease is not statistically significant (top1::OH1/2/3, bottom1, P > 0.05). The presence of a G-quadruplex significantly increases ΔG° (top1::GQ1/2, bottom1). Duplex destabilisation in the presence of a G-quadruplex is significant in the presence of 1 or 2 mismatches immediately proximal to the appended secondary structure motif (top1/top1::GQ2, bottom1-1MM and top1/top1::GQ2, bottom1-2MM). No significant changes are observable in the presence of three mismatches. Statistical significance of changes was estimated by analysis of variance, and Bonferroni correction for multiple testing was applied when necessary. **P < 0.01, ***P < 0.001.

Mentions: A number of thermal melting experiments were performed to determine whether and to what extent G-quadruplexes destabilize directly adjacent duplex DNA regions (25,26). Figures 2 and 3 show the results of the first set of sequences that were studied by UV melting experiments. The standard free enthalpy associated with annealing of the control duplex (top1, bottom1) was found to be −67.5 ± 1.7 kJ/mol with a melting temperature of 58.6 ± 0.7°C. The presence of a directly proximal G-quadruplex was observed to decrease the energy required for duplex melting by 14.8 to 16.8 kJ/mol, depending on the nature of the G-quadruplex (Table 2). For both sequence pairs, a smooth transition was observed on duplex melting, which is typical for a two-state transition (Supplementary Figures S1A and S2A). In accord with the experimental design depicted in Figure 1, duplex formation could be confirmed to be a bimolecular process, whereas G-quadruplex formation was unimolecular (Supplementary Figures S1D, S2D and F). CD experiments with top1/bottom1 and top1::GQ2/bottom provided evidence for G-quadruplex formation and minimal overlap between duplex and quadruplex melting (ΔTm >25°C, Supplementary Figure S2A and B). Linear dependence of spectral changes in the relevant temperature range and the results of van’t Hoff plot analysis further indicated that duplex dissociation is a cooperative process under the experimental conditions chosen for these studies (Supplementary Figures S1E, S2C and E).Figure 2.


Distance-dependent duplex DNA destabilization proximal to G-quadruplex/i-motif sequences.

König SL, Huppert JL, Sigel RK, Evans AC - Nucleic Acids Res. (2013)

Thermodynamic stability of a duplex derived from the c-kit promoter in the presence and in the absence of an overhang. In the absence of a 3′ overhang (top1, bottom1) ΔG° = −67.5 kJ/mol. A 3′-poly(T) overhang decreases thermodynamic stability (ΔG° = −65.0 kJ/mol), but this decrease is not statistically significant (top1::OH1/2/3, bottom1, P > 0.05). The presence of a G-quadruplex significantly increases ΔG° (top1::GQ1/2, bottom1). Duplex destabilisation in the presence of a G-quadruplex is significant in the presence of 1 or 2 mismatches immediately proximal to the appended secondary structure motif (top1/top1::GQ2, bottom1-1MM and top1/top1::GQ2, bottom1-2MM). No significant changes are observable in the presence of three mismatches. Statistical significance of changes was estimated by analysis of variance, and Bonferroni correction for multiple testing was applied when necessary. **P < 0.01, ***P < 0.001.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3753619&req=5

gkt476-F2: Thermodynamic stability of a duplex derived from the c-kit promoter in the presence and in the absence of an overhang. In the absence of a 3′ overhang (top1, bottom1) ΔG° = −67.5 kJ/mol. A 3′-poly(T) overhang decreases thermodynamic stability (ΔG° = −65.0 kJ/mol), but this decrease is not statistically significant (top1::OH1/2/3, bottom1, P > 0.05). The presence of a G-quadruplex significantly increases ΔG° (top1::GQ1/2, bottom1). Duplex destabilisation in the presence of a G-quadruplex is significant in the presence of 1 or 2 mismatches immediately proximal to the appended secondary structure motif (top1/top1::GQ2, bottom1-1MM and top1/top1::GQ2, bottom1-2MM). No significant changes are observable in the presence of three mismatches. Statistical significance of changes was estimated by analysis of variance, and Bonferroni correction for multiple testing was applied when necessary. **P < 0.01, ***P < 0.001.
Mentions: A number of thermal melting experiments were performed to determine whether and to what extent G-quadruplexes destabilize directly adjacent duplex DNA regions (25,26). Figures 2 and 3 show the results of the first set of sequences that were studied by UV melting experiments. The standard free enthalpy associated with annealing of the control duplex (top1, bottom1) was found to be −67.5 ± 1.7 kJ/mol with a melting temperature of 58.6 ± 0.7°C. The presence of a directly proximal G-quadruplex was observed to decrease the energy required for duplex melting by 14.8 to 16.8 kJ/mol, depending on the nature of the G-quadruplex (Table 2). For both sequence pairs, a smooth transition was observed on duplex melting, which is typical for a two-state transition (Supplementary Figures S1A and S2A). In accord with the experimental design depicted in Figure 1, duplex formation could be confirmed to be a bimolecular process, whereas G-quadruplex formation was unimolecular (Supplementary Figures S1D, S2D and F). CD experiments with top1/bottom1 and top1::GQ2/bottom provided evidence for G-quadruplex formation and minimal overlap between duplex and quadruplex melting (ΔTm >25°C, Supplementary Figure S2A and B). Linear dependence of spectral changes in the relevant temperature range and the results of van’t Hoff plot analysis further indicated that duplex dissociation is a cooperative process under the experimental conditions chosen for these studies (Supplementary Figures S1E, S2C and E).Figure 2.

Bottom Line: Prediction of putative G-quadruplex-forming regions is likely to be assisted by further understanding of what distance (number of base pairs) is required for duplexes to remain stable as quadruplexes or i-motifs form.Using oligonucleotide constructs derived from precedented G-quadruplexes and i-motif-forming bcl-2 P1 promoter region, initial biophysical stability studies indicate that the formation of G-quadruplex and i-motif conformations do destabilize proximal duplex regions.The undermining effect that quadruplex formation can have on duplex stability is mitigated with increased distance from the duplex region: a spacing of five base pairs or more is sufficient to maintain duplex stability proximal to predicted quadruplex/i-motif-forming regions.

View Article: PubMed Central - PubMed

Affiliation: Cavendish Laboratory, University of Cambridge, JJ Thomson Avenue, Cambridge CB3 0HE, UK, Institute of Inorganic Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland and University of Nice-Sophia Antipolis, UMR 7272 CNRS, Institut de 40 Chimie de Nice, 28 Avenue Valrose, 06108 Nice, France.

ABSTRACT
G-quadruplexes and i-motifs are complementary examples of non-canonical nucleic acid substructure conformations. G-quadruplex thermodynamic stability has been extensively studied for a variety of base sequences, but the degree of duplex destabilization that adjacent quadruplex structure formation can cause has yet to be fully addressed. Stable in vivo formation of these alternative nucleic acid structures is likely to be highly dependent on whether sufficient spacing exists between neighbouring duplex- and quadruplex-/i-motif-forming regions to accommodate quadruplexes or i-motifs without disrupting duplex stability. Prediction of putative G-quadruplex-forming regions is likely to be assisted by further understanding of what distance (number of base pairs) is required for duplexes to remain stable as quadruplexes or i-motifs form. Using oligonucleotide constructs derived from precedented G-quadruplexes and i-motif-forming bcl-2 P1 promoter region, initial biophysical stability studies indicate that the formation of G-quadruplex and i-motif conformations do destabilize proximal duplex regions. The undermining effect that quadruplex formation can have on duplex stability is mitigated with increased distance from the duplex region: a spacing of five base pairs or more is sufficient to maintain duplex stability proximal to predicted quadruplex/i-motif-forming regions.

Show MeSH
Related in: MedlinePlus