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Stability and kinetics of G-quadruplex structures.

Lane AN, Chaires JB, Gray RD, Trent JO - Nucleic Acids Res. (2008)

Bottom Line: Significant gaps in the literature have been identified, that should be filled by a systematic study of well-defined quadruplexes not only to provide the basic understanding of stability both for design purposes, but also as it relates to in vivo occurrence of quadruplexes.Quadruplex structures fold and unfold comparatively slowly, and DNA unwinding events associated with transcription and replication may be operating far from equilibrium.The kinetics of formation and resolution of quadruplexes, and methodologies are discussed in the context of stability and their possible biological occurrence.

View Article: PubMed Central - PubMed

Affiliation: Structural Biology Program, JG Brown Cancer Center, University of Louisville, KY 40202, USA. anlane01@gwise.louisville.edu

ABSTRACT
In this review, we give an overview of recent literature on the structure and stability of unimolecular G-rich quadruplex structures that are relevant to drug design and for in vivo function. The unifying theme in this review is energetics. The thermodynamic stability of quadruplexes has not been studied in the same detail as DNA and RNA duplexes, and there are important differences in the balance of forces between these classes of folded oligonucleotides. We provide an overview of the principles of stability and where available the experimental data that report on these principles. Significant gaps in the literature have been identified, that should be filled by a systematic study of well-defined quadruplexes not only to provide the basic understanding of stability both for design purposes, but also as it relates to in vivo occurrence of quadruplexes. Techniques that are commonly applied to the determination of the structure, stability and folding are discussed in terms of information content and limitations. Quadruplex structures fold and unfold comparatively slowly, and DNA unwinding events associated with transcription and replication may be operating far from equilibrium. The kinetics of formation and resolution of quadruplexes, and methodologies are discussed in the context of stability and their possible biological occurrence.

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Thermal profiles for two folding pathways. The populations of states in two possible pathways as described in the text was calculated. Model (i) Two species connected by unfolded state: N1⇔D + N2 ⇔ D. The reference temperature, Tref = 273 K. The equilibrium constant K0 for unfolding at 273 K = 1E − 5. The unfolding enthalpies were Δ H1 (N1) = 40 kcal mol−1, Δ H2(N2) = 60 kcal mol−1. For these parameters, Tm1 = 324 K Tm2 = 305 K. (A) Populations as a function of temperature. Red square: state D; open black circles: state N2; filled blue circles: state N1. (B) Changes in absorbance as a function of temperature for εN1 = εN2 (filled red squares), εN1 = 1.2, εN2 = 1 (open blue squares) and εN1 = 1, εN2 = 1.2 (open black circles). Best fit to a single transition with εN1 = εN2: ΔH = 40 kcal mol−1; K0 = 9.7 E−6 at 273 K; Tm = 324 K. Model (ii) sequential unfolding N ⇔ K1I ⇔ K2K1 = K2 = 1E−5 at 273 K, Δ H1 = 30 kcal mol−1, Δ H2 = 20 kcal mol−1. For these parameters, Tm1 = 305 K, Tm2 = 324 K. (C) Populations of N (red squares), I (black circles) and U (blue squares). The populations of N and I are equal at 305 K, and N is dominant at low temperature. (D) Thermal melting profile using the populations in C, and difference absorption coefficients of (a) ΔεN = ΔεI = 1 (blue squares) (b) ΔεN = 1.2 ΔεI = 1 (black circles); (c) ΔεN = 1 ΔεI = 1.2 (blue squares). The fits for conditions a and b to a single folding transition are shown as thin continuous lines, and the recovered parameter values were: (A) K(273) = 8.7 E−6, ΔH = 40.5 ± 0.07 kcal mol−1, Δε = 1.0, R2 = 0.99997. (B) K(273) = 1.68 E−4; ΔH = 31.6 ± 0.3 kcal mol−1, Δε = 1.19, R2 = 0.99895. Parameter estimates are thus unreliable if the wrong model is used, even where the data appear as a simple transition.
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Figure 8: Thermal profiles for two folding pathways. The populations of states in two possible pathways as described in the text was calculated. Model (i) Two species connected by unfolded state: N1⇔D + N2 ⇔ D. The reference temperature, Tref = 273 K. The equilibrium constant K0 for unfolding at 273 K = 1E − 5. The unfolding enthalpies were Δ H1 (N1) = 40 kcal mol−1, Δ H2(N2) = 60 kcal mol−1. For these parameters, Tm1 = 324 K Tm2 = 305 K. (A) Populations as a function of temperature. Red square: state D; open black circles: state N2; filled blue circles: state N1. (B) Changes in absorbance as a function of temperature for εN1 = εN2 (filled red squares), εN1 = 1.2, εN2 = 1 (open blue squares) and εN1 = 1, εN2 = 1.2 (open black circles). Best fit to a single transition with εN1 = εN2: ΔH = 40 kcal mol−1; K0 = 9.7 E−6 at 273 K; Tm = 324 K. Model (ii) sequential unfolding N ⇔ K1I ⇔ K2K1 = K2 = 1E−5 at 273 K, Δ H1 = 30 kcal mol−1, Δ H2 = 20 kcal mol−1. For these parameters, Tm1 = 305 K, Tm2 = 324 K. (C) Populations of N (red squares), I (black circles) and U (blue squares). The populations of N and I are equal at 305 K, and N is dominant at low temperature. (D) Thermal melting profile using the populations in C, and difference absorption coefficients of (a) ΔεN = ΔεI = 1 (blue squares) (b) ΔεN = 1.2 ΔεI = 1 (black circles); (c) ΔεN = 1 ΔεI = 1.2 (blue squares). The fits for conditions a and b to a single folding transition are shown as thin continuous lines, and the recovered parameter values were: (A) K(273) = 8.7 E−6, ΔH = 40.5 ± 0.07 kcal mol−1, Δε = 1.0, R2 = 0.99997. (B) K(273) = 1.68 E−4; ΔH = 31.6 ± 0.3 kcal mol−1, Δε = 1.19, R2 = 0.99895. Parameter estimates are thus unreliable if the wrong model is used, even where the data appear as a simple transition.

Mentions: These models differ in that the initial (native) points have in principle distinguishable properties. Model 1 starts with a mixture of states that independently evolve toward the common end state, whereas model 2 implies that the two states present at low T (i.e. N and I, populations determined by K2 at sufficiently low temperature for example) evolves through the intermediate state I. This is why although the same number of states is involved in the two mechanisms, the unfolding behavior may be different depending on the specific values of the equilibrium constants, their enthalpy differences and the values of Δσ. Figure 8 shows a simple simulated comparison of an optical unfolding experiment in which there is an unfolded ensemble at high temperature, and a folded ensemble at low temperature. The two models differ in the folded ensemble. The first model posits two alternative conformations that do not interconvert directly on any realistic experimental timescale (they interconvert exclusively through the unfolded state). The second model posits a sequential pathway with a fully native fold proceeding to the unfolded ensemble via an obligatory intermediate. The relative populations of N1 and N2 are determined by the ratio K2/K1. In this simulation, these have been made equal at low temperature. At low T, the distributions of I and N are determined by the equilibrium constant between them. Depending on the assumptions, the denaturation curve may appear either monophasic or biphasic. The simulated curves were fitted to the equation for a single transition, i.e. three parameters, assuming that the heat capacity difference between states is zero. All curves in this instance are well represented by a single transition, but the recovered enthalpies did not match the input values. For biphasic curves, of course one would fit a two-state transition of some kind for which the parameters would be better defined, assuming the correct model were chosen (sic).Figure 8.


Stability and kinetics of G-quadruplex structures.

Lane AN, Chaires JB, Gray RD, Trent JO - Nucleic Acids Res. (2008)

Thermal profiles for two folding pathways. The populations of states in two possible pathways as described in the text was calculated. Model (i) Two species connected by unfolded state: N1⇔D + N2 ⇔ D. The reference temperature, Tref = 273 K. The equilibrium constant K0 for unfolding at 273 K = 1E − 5. The unfolding enthalpies were Δ H1 (N1) = 40 kcal mol−1, Δ H2(N2) = 60 kcal mol−1. For these parameters, Tm1 = 324 K Tm2 = 305 K. (A) Populations as a function of temperature. Red square: state D; open black circles: state N2; filled blue circles: state N1. (B) Changes in absorbance as a function of temperature for εN1 = εN2 (filled red squares), εN1 = 1.2, εN2 = 1 (open blue squares) and εN1 = 1, εN2 = 1.2 (open black circles). Best fit to a single transition with εN1 = εN2: ΔH = 40 kcal mol−1; K0 = 9.7 E−6 at 273 K; Tm = 324 K. Model (ii) sequential unfolding N ⇔ K1I ⇔ K2K1 = K2 = 1E−5 at 273 K, Δ H1 = 30 kcal mol−1, Δ H2 = 20 kcal mol−1. For these parameters, Tm1 = 305 K, Tm2 = 324 K. (C) Populations of N (red squares), I (black circles) and U (blue squares). The populations of N and I are equal at 305 K, and N is dominant at low temperature. (D) Thermal melting profile using the populations in C, and difference absorption coefficients of (a) ΔεN = ΔεI = 1 (blue squares) (b) ΔεN = 1.2 ΔεI = 1 (black circles); (c) ΔεN = 1 ΔεI = 1.2 (blue squares). The fits for conditions a and b to a single folding transition are shown as thin continuous lines, and the recovered parameter values were: (A) K(273) = 8.7 E−6, ΔH = 40.5 ± 0.07 kcal mol−1, Δε = 1.0, R2 = 0.99997. (B) K(273) = 1.68 E−4; ΔH = 31.6 ± 0.3 kcal mol−1, Δε = 1.19, R2 = 0.99895. Parameter estimates are thus unreliable if the wrong model is used, even where the data appear as a simple transition.
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Figure 8: Thermal profiles for two folding pathways. The populations of states in two possible pathways as described in the text was calculated. Model (i) Two species connected by unfolded state: N1⇔D + N2 ⇔ D. The reference temperature, Tref = 273 K. The equilibrium constant K0 for unfolding at 273 K = 1E − 5. The unfolding enthalpies were Δ H1 (N1) = 40 kcal mol−1, Δ H2(N2) = 60 kcal mol−1. For these parameters, Tm1 = 324 K Tm2 = 305 K. (A) Populations as a function of temperature. Red square: state D; open black circles: state N2; filled blue circles: state N1. (B) Changes in absorbance as a function of temperature for εN1 = εN2 (filled red squares), εN1 = 1.2, εN2 = 1 (open blue squares) and εN1 = 1, εN2 = 1.2 (open black circles). Best fit to a single transition with εN1 = εN2: ΔH = 40 kcal mol−1; K0 = 9.7 E−6 at 273 K; Tm = 324 K. Model (ii) sequential unfolding N ⇔ K1I ⇔ K2K1 = K2 = 1E−5 at 273 K, Δ H1 = 30 kcal mol−1, Δ H2 = 20 kcal mol−1. For these parameters, Tm1 = 305 K, Tm2 = 324 K. (C) Populations of N (red squares), I (black circles) and U (blue squares). The populations of N and I are equal at 305 K, and N is dominant at low temperature. (D) Thermal melting profile using the populations in C, and difference absorption coefficients of (a) ΔεN = ΔεI = 1 (blue squares) (b) ΔεN = 1.2 ΔεI = 1 (black circles); (c) ΔεN = 1 ΔεI = 1.2 (blue squares). The fits for conditions a and b to a single folding transition are shown as thin continuous lines, and the recovered parameter values were: (A) K(273) = 8.7 E−6, ΔH = 40.5 ± 0.07 kcal mol−1, Δε = 1.0, R2 = 0.99997. (B) K(273) = 1.68 E−4; ΔH = 31.6 ± 0.3 kcal mol−1, Δε = 1.19, R2 = 0.99895. Parameter estimates are thus unreliable if the wrong model is used, even where the data appear as a simple transition.
Mentions: These models differ in that the initial (native) points have in principle distinguishable properties. Model 1 starts with a mixture of states that independently evolve toward the common end state, whereas model 2 implies that the two states present at low T (i.e. N and I, populations determined by K2 at sufficiently low temperature for example) evolves through the intermediate state I. This is why although the same number of states is involved in the two mechanisms, the unfolding behavior may be different depending on the specific values of the equilibrium constants, their enthalpy differences and the values of Δσ. Figure 8 shows a simple simulated comparison of an optical unfolding experiment in which there is an unfolded ensemble at high temperature, and a folded ensemble at low temperature. The two models differ in the folded ensemble. The first model posits two alternative conformations that do not interconvert directly on any realistic experimental timescale (they interconvert exclusively through the unfolded state). The second model posits a sequential pathway with a fully native fold proceeding to the unfolded ensemble via an obligatory intermediate. The relative populations of N1 and N2 are determined by the ratio K2/K1. In this simulation, these have been made equal at low temperature. At low T, the distributions of I and N are determined by the equilibrium constant between them. Depending on the assumptions, the denaturation curve may appear either monophasic or biphasic. The simulated curves were fitted to the equation for a single transition, i.e. three parameters, assuming that the heat capacity difference between states is zero. All curves in this instance are well represented by a single transition, but the recovered enthalpies did not match the input values. For biphasic curves, of course one would fit a two-state transition of some kind for which the parameters would be better defined, assuming the correct model were chosen (sic).Figure 8.

Bottom Line: Significant gaps in the literature have been identified, that should be filled by a systematic study of well-defined quadruplexes not only to provide the basic understanding of stability both for design purposes, but also as it relates to in vivo occurrence of quadruplexes.Quadruplex structures fold and unfold comparatively slowly, and DNA unwinding events associated with transcription and replication may be operating far from equilibrium.The kinetics of formation and resolution of quadruplexes, and methodologies are discussed in the context of stability and their possible biological occurrence.

View Article: PubMed Central - PubMed

Affiliation: Structural Biology Program, JG Brown Cancer Center, University of Louisville, KY 40202, USA. anlane01@gwise.louisville.edu

ABSTRACT
In this review, we give an overview of recent literature on the structure and stability of unimolecular G-rich quadruplex structures that are relevant to drug design and for in vivo function. The unifying theme in this review is energetics. The thermodynamic stability of quadruplexes has not been studied in the same detail as DNA and RNA duplexes, and there are important differences in the balance of forces between these classes of folded oligonucleotides. We provide an overview of the principles of stability and where available the experimental data that report on these principles. Significant gaps in the literature have been identified, that should be filled by a systematic study of well-defined quadruplexes not only to provide the basic understanding of stability both for design purposes, but also as it relates to in vivo occurrence of quadruplexes. Techniques that are commonly applied to the determination of the structure, stability and folding are discussed in terms of information content and limitations. Quadruplex structures fold and unfold comparatively slowly, and DNA unwinding events associated with transcription and replication may be operating far from equilibrium. The kinetics of formation and resolution of quadruplexes, and methodologies are discussed in the context of stability and their possible biological occurrence.

Show MeSH
Related in: MedlinePlus