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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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G-quadruplex-induced duplex destabilization of a modified c-kit fragment. A G-quadruplex strongly destabilizes the adjacent duplex (top2::GQ2, bottom2). An increasing number of mismatches proximal to the G-quadruplex region results in a significant decrease in ΔΔG° (top2::GQ2, bottom2-1-5MM), approximating zero in the case of six mismatches (top2::GQ2, bottom2-6MM). *P < 0.05, ***P < 0.001.
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gkt476-F4: G-quadruplex-induced duplex destabilization of a modified c-kit fragment. A G-quadruplex strongly destabilizes the adjacent duplex (top2::GQ2, bottom2). An increasing number of mismatches proximal to the G-quadruplex region results in a significant decrease in ΔΔG° (top2::GQ2, bottom2-1-5MM), approximating zero in the case of six mismatches (top2::GQ2, bottom2-6MM). *P < 0.05, ***P < 0.001.

Mentions: Similar observations were made for a second set of sequences derived from a modified version of the c-kit promoter fragment (Figure 4). The presence of the Tetrahymena telomeric G-quadruplex was found to decrease duplex stability by 15.2 kJ/mol (Table 2). As the number of mismatches was increased, duplex ΔΔG° was found to decrease in a regular manner [ΔΔG°(top2::GQ2, bottom2-1MM) = 13.1 ± 2.6 kJ/mol; ΔΔG°(top2::GQ2, bottom2-2MM) = 10.9 ± 2.4 kJ/mol; ΔΔG°(top2::GQ2, bottom2-3MM) = 7.2 ± 2.2 kJ/mol; ΔΔG°(top2::GQ2, bottom2-4MM) = 5.1 ± 1.4 kJ/mol; ΔΔG°(top2::GQ2, bottom2-5MM) = 3.0 ± 1.9 kJ/mol]. On insertion of six mismatches, ΔΔG° was found to approximate zero and become statistically insignificant (top2::GQ2, bottom2-6MM). Statistical analysis was performed in an analogous manner as for the first set of sequences. Again, the Tetrahymena G-quadruplex displayed high thermostability (Tm ∼80°C, ΔTm > 25°C) and was shown to be unimolecular, whereas duplex interaction was demonstrated to be an intermolecular process that can be described by a two-state model (Supplementary Figure S3). The results of the second series of melting experiments are summarized in the Supplementary Information (Supplementary Table S3).Figure 4.


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

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

G-quadruplex-induced duplex destabilization of a modified c-kit fragment. A G-quadruplex strongly destabilizes the adjacent duplex (top2::GQ2, bottom2). An increasing number of mismatches proximal to the G-quadruplex region results in a significant decrease in ΔΔG° (top2::GQ2, bottom2-1-5MM), approximating zero in the case of six mismatches (top2::GQ2, bottom2-6MM). *P < 0.05, ***P < 0.001.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt476-F4: G-quadruplex-induced duplex destabilization of a modified c-kit fragment. A G-quadruplex strongly destabilizes the adjacent duplex (top2::GQ2, bottom2). An increasing number of mismatches proximal to the G-quadruplex region results in a significant decrease in ΔΔG° (top2::GQ2, bottom2-1-5MM), approximating zero in the case of six mismatches (top2::GQ2, bottom2-6MM). *P < 0.05, ***P < 0.001.
Mentions: Similar observations were made for a second set of sequences derived from a modified version of the c-kit promoter fragment (Figure 4). The presence of the Tetrahymena telomeric G-quadruplex was found to decrease duplex stability by 15.2 kJ/mol (Table 2). As the number of mismatches was increased, duplex ΔΔG° was found to decrease in a regular manner [ΔΔG°(top2::GQ2, bottom2-1MM) = 13.1 ± 2.6 kJ/mol; ΔΔG°(top2::GQ2, bottom2-2MM) = 10.9 ± 2.4 kJ/mol; ΔΔG°(top2::GQ2, bottom2-3MM) = 7.2 ± 2.2 kJ/mol; ΔΔG°(top2::GQ2, bottom2-4MM) = 5.1 ± 1.4 kJ/mol; ΔΔG°(top2::GQ2, bottom2-5MM) = 3.0 ± 1.9 kJ/mol]. On insertion of six mismatches, ΔΔG° was found to approximate zero and become statistically insignificant (top2::GQ2, bottom2-6MM). Statistical analysis was performed in an analogous manner as for the first set of sequences. Again, the Tetrahymena G-quadruplex displayed high thermostability (Tm ∼80°C, ΔTm > 25°C) and was shown to be unimolecular, whereas duplex interaction was demonstrated to be an intermolecular process that can be described by a two-state model (Supplementary Figure S3). The results of the second series of melting experiments are summarized in the Supplementary Information (Supplementary Table S3).Figure 4.

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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