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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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i-motif-induced duplex destabilization of a modified c-kit fragment. The i-motif destabilizes the neighbouring double helix (top2::IM, bottom2). Increasing the number of mismatches proximal to the i-motif results in significantly decreasing ΔΔG°, approximating zero on introducing three mismatches at the vicinity to the i-motif (top2::IM, bottom2-3MM). *P < 0.05, ***P < 0.001.
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gkt476-F5: i-motif-induced duplex destabilization of a modified c-kit fragment. The i-motif destabilizes the neighbouring double helix (top2::IM, bottom2). Increasing the number of mismatches proximal to the i-motif results in significantly decreasing ΔΔG°, approximating zero on introducing three mismatches at the vicinity to the i-motif (top2::IM, bottom2-3MM). *P < 0.05, ***P < 0.001.

Mentions: To determine whether formation of a proximal i-motif induces destabilization of an adjacent duplex stretch, an additional series of UV melting experiments were performed (Figure 5). Owing to the pH sensitivity and to avoid overlap of duplex and i-motif unfolding, these experiments were carried out at pH 4.0 and 4.4, respectively. Attachment of the known IM sequence Py39WT to the 3′-end of C-1B was found to significantly decrease the stability of the adjacent duplex by 6.0 kJ/mol (Table 2). As mismatches were introduced proximal to the i-motif-forming region, ΔΔG was found to progressively decrease [ΔΔG° (top2::IM, bottom2-1MM) = 4.3 ± 1.2 kJ/mol; ΔΔG°(top2::IM, bottom2-2MM) = 3.3 ± 1.0 kJ/mol]. After three mismatches had been introduced, ΔΔG° approximated zero (top2::IM, bottom2-3MM). In all cases, melting of the i-motif was found to occur at significantly higher temperatures than duplex denaturation (ΔTm > 30°C), and the altered overhang did not seem to affect the unfolding mechanism of the duplex, as it could again be shown to be an intermolecular process that could be approximated by a two-state model (Supplementary Figure S4). All results are summarized in the Supplementary Information (Supplementary Table S4).Figure 5.


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

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

i-motif-induced duplex destabilization of a modified c-kit fragment. The i-motif destabilizes the neighbouring double helix (top2::IM, bottom2). Increasing the number of mismatches proximal to the i-motif results in significantly decreasing ΔΔG°, approximating zero on introducing three mismatches at the vicinity to the i-motif (top2::IM, bottom2-3MM). *P < 0.05, ***P < 0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3753619&req=5

gkt476-F5: i-motif-induced duplex destabilization of a modified c-kit fragment. The i-motif destabilizes the neighbouring double helix (top2::IM, bottom2). Increasing the number of mismatches proximal to the i-motif results in significantly decreasing ΔΔG°, approximating zero on introducing three mismatches at the vicinity to the i-motif (top2::IM, bottom2-3MM). *P < 0.05, ***P < 0.001.
Mentions: To determine whether formation of a proximal i-motif induces destabilization of an adjacent duplex stretch, an additional series of UV melting experiments were performed (Figure 5). Owing to the pH sensitivity and to avoid overlap of duplex and i-motif unfolding, these experiments were carried out at pH 4.0 and 4.4, respectively. Attachment of the known IM sequence Py39WT to the 3′-end of C-1B was found to significantly decrease the stability of the adjacent duplex by 6.0 kJ/mol (Table 2). As mismatches were introduced proximal to the i-motif-forming region, ΔΔG was found to progressively decrease [ΔΔG° (top2::IM, bottom2-1MM) = 4.3 ± 1.2 kJ/mol; ΔΔG°(top2::IM, bottom2-2MM) = 3.3 ± 1.0 kJ/mol]. After three mismatches had been introduced, ΔΔG° approximated zero (top2::IM, bottom2-3MM). In all cases, melting of the i-motif was found to occur at significantly higher temperatures than duplex denaturation (ΔTm > 30°C), and the altered overhang did not seem to affect the unfolding mechanism of the duplex, as it could again be shown to be an intermolecular process that could be approximated by a two-state model (Supplementary Figure S4). All results are summarized in the Supplementary Information (Supplementary Table S4).Figure 5.

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