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Single molecule conformational analysis of DNA G-quadruplexes.

Shirude PS, Balasubramanian S - Biochimie (2008)

Bottom Line: Single molecule fluorescence resonance energy transfer (FRET) can be employed to study conformational heterogeneity and real-time dynamics of biological macromolecules.Here we present single molecule studies on human genomic DNA G-quadruplex sequences that occur in the telomeres and in the promoter of a proto-oncogene.The findings are discussed with respect to the proposed biological function(s) of such motifs in living cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK.

ABSTRACT
Single molecule fluorescence resonance energy transfer (FRET) can be employed to study conformational heterogeneity and real-time dynamics of biological macromolecules. Here we present single molecule studies on human genomic DNA G-quadruplex sequences that occur in the telomeres and in the promoter of a proto-oncogene. The findings are discussed with respect to the proposed biological function(s) of such motifs in living cells.

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(A) Changes in single-molecule FRET histograms during the opening of the DNA quadruplex by hybridization to 7 nM III. The ‘zero’ peak at t = 0 has been subtracted from all histograms for clarity. The experiment was carried out at 20 °C in 100 mM NaCl and 10 mM Tris–HCl (pH 7.4). (B) Kinetic traces for the low- and high-FRET subpopulations. Data were fit to a single exponential. Copyright (2003) National Academy of Sciences of the United States of America [57].
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fig5: (A) Changes in single-molecule FRET histograms during the opening of the DNA quadruplex by hybridization to 7 nM III. The ‘zero’ peak at t = 0 has been subtracted from all histograms for clarity. The experiment was carried out at 20 °C in 100 mM NaCl and 10 mM Tris–HCl (pH 7.4). (B) Kinetic traces for the low- and high-FRET subpopulations. Data were fit to a single exponential. Copyright (2003) National Academy of Sciences of the United States of America [57].

Mentions: Ying et al. demonstrated [57] that two conformations X and Y (Fig. 3) apparently unfold at the same rate in a second-order hybridization reaction (Fig. 5). In the presence of potassium, the activation free energy of unfolding (ΔGactivation = 22.6 kcal mol−1 at 37 °C) was largely entropic (ΔHactivation = 6.4 ± 0.4 kcal mol−1, ΔSactivation = −52.3 ± 1.4 cal mol−1 K−1), suggesting that the transition state is pre-organized in a favourable conformation for hybridization to occur. However, in the presence of sodium ions, although the free energy of activation (ΔGactivation = 22.3 kcal mol−1 at 37 °C) was similar, a relatively larger enthalpic contribution was observed (ΔHactivation = 14.9 ± 0.2 kcal mol−1, ΔSactivation = −23.0 ± 0.8 cal mol−1 K−1). Therefore it was hypothesized that different transition-state structures exist depending on the presence of either sodium or potassium ions. The analysis of single-molecule dynamics by Lee et al. [58] revealed transitions between all three FRET states (U, F1, and F2), most frequently in 2 mM K+ (Fig. 6B). Most of the transitions between F1 and F2 passed through U. A direct transition between two folded conformations without unfolding is highly unlikely considering the folded topology of G-quadruplexes. According to the analysis of dwell times, they further classified the molecular conformations into either long-lived species, if the dwell time was >100 s and short-lived species if the dwell time was <100 s. In total, six distinct states were observed: long-lived states LU, LF1, and LF2, and short-lived states SU, SF1, and SF2 (Fig. 6A). The long-lived folded states, LF1 and LF2 (Fig. 6A), were dominant in high K+ concentrations and were assigned to the parallel and antiparallel structures respectively, based on comparison with NMR spectroscopic studies of temperature dependence [79]. The long-lived unfolded state, LU, was highly populated at low K+ concentrations and displayed the characteristic salt dependence of a disordered ssDNA. SF1 and SF2 were much less stable than their long-lived counterparts. SU was clearly distinct from LU in having a much shorter lifetime. Although SF1 and SF2 differed from LF1 and LF2 in microscopic detail, their similar FRET values suggested that they may have the same global folds.


Single molecule conformational analysis of DNA G-quadruplexes.

Shirude PS, Balasubramanian S - Biochimie (2008)

(A) Changes in single-molecule FRET histograms during the opening of the DNA quadruplex by hybridization to 7 nM III. The ‘zero’ peak at t = 0 has been subtracted from all histograms for clarity. The experiment was carried out at 20 °C in 100 mM NaCl and 10 mM Tris–HCl (pH 7.4). (B) Kinetic traces for the low- and high-FRET subpopulations. Data were fit to a single exponential. Copyright (2003) National Academy of Sciences of the United States of America [57].
© Copyright Policy
Related In: Results  -  Collection

License
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fig5: (A) Changes in single-molecule FRET histograms during the opening of the DNA quadruplex by hybridization to 7 nM III. The ‘zero’ peak at t = 0 has been subtracted from all histograms for clarity. The experiment was carried out at 20 °C in 100 mM NaCl and 10 mM Tris–HCl (pH 7.4). (B) Kinetic traces for the low- and high-FRET subpopulations. Data were fit to a single exponential. Copyright (2003) National Academy of Sciences of the United States of America [57].
Mentions: Ying et al. demonstrated [57] that two conformations X and Y (Fig. 3) apparently unfold at the same rate in a second-order hybridization reaction (Fig. 5). In the presence of potassium, the activation free energy of unfolding (ΔGactivation = 22.6 kcal mol−1 at 37 °C) was largely entropic (ΔHactivation = 6.4 ± 0.4 kcal mol−1, ΔSactivation = −52.3 ± 1.4 cal mol−1 K−1), suggesting that the transition state is pre-organized in a favourable conformation for hybridization to occur. However, in the presence of sodium ions, although the free energy of activation (ΔGactivation = 22.3 kcal mol−1 at 37 °C) was similar, a relatively larger enthalpic contribution was observed (ΔHactivation = 14.9 ± 0.2 kcal mol−1, ΔSactivation = −23.0 ± 0.8 cal mol−1 K−1). Therefore it was hypothesized that different transition-state structures exist depending on the presence of either sodium or potassium ions. The analysis of single-molecule dynamics by Lee et al. [58] revealed transitions between all three FRET states (U, F1, and F2), most frequently in 2 mM K+ (Fig. 6B). Most of the transitions between F1 and F2 passed through U. A direct transition between two folded conformations without unfolding is highly unlikely considering the folded topology of G-quadruplexes. According to the analysis of dwell times, they further classified the molecular conformations into either long-lived species, if the dwell time was >100 s and short-lived species if the dwell time was <100 s. In total, six distinct states were observed: long-lived states LU, LF1, and LF2, and short-lived states SU, SF1, and SF2 (Fig. 6A). The long-lived folded states, LF1 and LF2 (Fig. 6A), were dominant in high K+ concentrations and were assigned to the parallel and antiparallel structures respectively, based on comparison with NMR spectroscopic studies of temperature dependence [79]. The long-lived unfolded state, LU, was highly populated at low K+ concentrations and displayed the characteristic salt dependence of a disordered ssDNA. SF1 and SF2 were much less stable than their long-lived counterparts. SU was clearly distinct from LU in having a much shorter lifetime. Although SF1 and SF2 differed from LF1 and LF2 in microscopic detail, their similar FRET values suggested that they may have the same global folds.

Bottom Line: Single molecule fluorescence resonance energy transfer (FRET) can be employed to study conformational heterogeneity and real-time dynamics of biological macromolecules.Here we present single molecule studies on human genomic DNA G-quadruplex sequences that occur in the telomeres and in the promoter of a proto-oncogene.The findings are discussed with respect to the proposed biological function(s) of such motifs in living cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK.

ABSTRACT
Single molecule fluorescence resonance energy transfer (FRET) can be employed to study conformational heterogeneity and real-time dynamics of biological macromolecules. Here we present single molecule studies on human genomic DNA G-quadruplex sequences that occur in the telomeres and in the promoter of a proto-oncogene. The findings are discussed with respect to the proposed biological function(s) of such motifs in living cells.

Show MeSH