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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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Schemes of the four-stranded structures in the focus of this study and experimental design. (A) Schematic of a G-quadruplex. Poly(G) stretches interact via W–C and Hoogsteen hydrogen bonds to form G-tetrads (squares) capable of stacking together. Quadruplex thermodynamic stability is enhanced by mono- and divalent cations (grey spheres). (B) Schematic of an i-motif: Poly(C) pairs interact in protic environments (squares) and stack together in an intercalated manner. (C) Characterization of duplex stability in various molecular environments. References: Two different double-strands (top1, bottom1 and top2, bottom2) are denatured when they are subjected to high temperature and re-associate at decreasing temperature. Mismatches (MM) decrease thermal stability and are introduced to induce structural flexibility and one end of the duplex. G-quadruplex, i-motif: Thermal melting with two different G-quadruplex-forming sequences (GQ1 and GQ2) and an IM sequence directly joined to one of the duplex strands. The thermodynamic parameters associated with duplex dissociation were subsequently compared with the reference. Control: Poly(T) single-stranded overhangs of varying length (poly(T)1, poly(T)2, poly(T)3) are appended to the duplex to characterize the effect of an unstructured overhang on duplex thermodynamics.
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gkt476-F1: Schemes of the four-stranded structures in the focus of this study and experimental design. (A) Schematic of a G-quadruplex. Poly(G) stretches interact via W–C and Hoogsteen hydrogen bonds to form G-tetrads (squares) capable of stacking together. Quadruplex thermodynamic stability is enhanced by mono- and divalent cations (grey spheres). (B) Schematic of an i-motif: Poly(C) pairs interact in protic environments (squares) and stack together in an intercalated manner. (C) Characterization of duplex stability in various molecular environments. References: Two different double-strands (top1, bottom1 and top2, bottom2) are denatured when they are subjected to high temperature and re-associate at decreasing temperature. Mismatches (MM) decrease thermal stability and are introduced to induce structural flexibility and one end of the duplex. G-quadruplex, i-motif: Thermal melting with two different G-quadruplex-forming sequences (GQ1 and GQ2) and an IM sequence directly joined to one of the duplex strands. The thermodynamic parameters associated with duplex dissociation were subsequently compared with the reference. Control: Poly(T) single-stranded overhangs of varying length (poly(T)1, poly(T)2, poly(T)3) are appended to the duplex to characterize the effect of an unstructured overhang on duplex thermodynamics.

Mentions: The canonical Watson–Crick (W–C) double-helix conformation of DNA has been extensively studied and is well understood. It is less well known that nucleic acids can also form alternative conformational structures, such as G-quadruplexes and i-motifs. A G-quadruplex conformer consists of stacks of G-quartets, which are composed of four guanine bases arranged in a plane and stabilised by Hoogsteen (H) and W–C hydrogen-bonding interactions (1). Enhanced electrostatic and π–π-bonding interactions cause these G-quartets to stack together into G-quadruplex conformers: this stacking has been found to be strongly favoured by the presence of mono- and divalent cations (Figure 1A) (2,3). A wide variety of G-quadruplex topologies have been demonstrated to exist, each varying with regard to the number of guanines involved, relative strand orientation and loop topology (4,5). G-quadruplex stability in isolation has been thoroughly studied, and significant influencing factors include the number of stacked G-quartets, G-quadruplex topology and the type of G-quadruplex-binding cation present in solution (6–11). A G-quadruplex is most likely to form in the presence of at least four consecutive G-runs; hence, putative G-quadruplex-forming regions can be predicted by scanning through nucleic acid genetic sequences (12).Figure 1.


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

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

Schemes of the four-stranded structures in the focus of this study and experimental design. (A) Schematic of a G-quadruplex. Poly(G) stretches interact via W–C and Hoogsteen hydrogen bonds to form G-tetrads (squares) capable of stacking together. Quadruplex thermodynamic stability is enhanced by mono- and divalent cations (grey spheres). (B) Schematic of an i-motif: Poly(C) pairs interact in protic environments (squares) and stack together in an intercalated manner. (C) Characterization of duplex stability in various molecular environments. References: Two different double-strands (top1, bottom1 and top2, bottom2) are denatured when they are subjected to high temperature and re-associate at decreasing temperature. Mismatches (MM) decrease thermal stability and are introduced to induce structural flexibility and one end of the duplex. G-quadruplex, i-motif: Thermal melting with two different G-quadruplex-forming sequences (GQ1 and GQ2) and an IM sequence directly joined to one of the duplex strands. The thermodynamic parameters associated with duplex dissociation were subsequently compared with the reference. Control: Poly(T) single-stranded overhangs of varying length (poly(T)1, poly(T)2, poly(T)3) are appended to the duplex to characterize the effect of an unstructured overhang on duplex thermodynamics.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt476-F1: Schemes of the four-stranded structures in the focus of this study and experimental design. (A) Schematic of a G-quadruplex. Poly(G) stretches interact via W–C and Hoogsteen hydrogen bonds to form G-tetrads (squares) capable of stacking together. Quadruplex thermodynamic stability is enhanced by mono- and divalent cations (grey spheres). (B) Schematic of an i-motif: Poly(C) pairs interact in protic environments (squares) and stack together in an intercalated manner. (C) Characterization of duplex stability in various molecular environments. References: Two different double-strands (top1, bottom1 and top2, bottom2) are denatured when they are subjected to high temperature and re-associate at decreasing temperature. Mismatches (MM) decrease thermal stability and are introduced to induce structural flexibility and one end of the duplex. G-quadruplex, i-motif: Thermal melting with two different G-quadruplex-forming sequences (GQ1 and GQ2) and an IM sequence directly joined to one of the duplex strands. The thermodynamic parameters associated with duplex dissociation were subsequently compared with the reference. Control: Poly(T) single-stranded overhangs of varying length (poly(T)1, poly(T)2, poly(T)3) are appended to the duplex to characterize the effect of an unstructured overhang on duplex thermodynamics.
Mentions: The canonical Watson–Crick (W–C) double-helix conformation of DNA has been extensively studied and is well understood. It is less well known that nucleic acids can also form alternative conformational structures, such as G-quadruplexes and i-motifs. A G-quadruplex conformer consists of stacks of G-quartets, which are composed of four guanine bases arranged in a plane and stabilised by Hoogsteen (H) and W–C hydrogen-bonding interactions (1). Enhanced electrostatic and π–π-bonding interactions cause these G-quartets to stack together into G-quadruplex conformers: this stacking has been found to be strongly favoured by the presence of mono- and divalent cations (Figure 1A) (2,3). A wide variety of G-quadruplex topologies have been demonstrated to exist, each varying with regard to the number of guanines involved, relative strand orientation and loop topology (4,5). G-quadruplex stability in isolation has been thoroughly studied, and significant influencing factors include the number of stacked G-quartets, G-quadruplex topology and the type of G-quadruplex-binding cation present in solution (6–11). A G-quadruplex is most likely to form in the presence of at least four consecutive G-runs; hence, putative G-quadruplex-forming regions can be predicted by scanning through nucleic acid genetic sequences (12).Figure 1.

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