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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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Absorbance and CD spectra. UV absorbance (A) and circular dichroic (B) spectra of the human telomere quadruplex sequence 5′AGGG(TTAGGG)3 in phosphate buffer (pH 7.0) containing 200 mM NaCl. Spectra obtained at 20°C are indicated by the solid line and correspond to the fully folded quadruplex form. Spectra obtained at 95°C are indicated by the dotted line, and correspond to the denatured, unfolded form.
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Figure 5: Absorbance and CD spectra. UV absorbance (A) and circular dichroic (B) spectra of the human telomere quadruplex sequence 5′AGGG(TTAGGG)3 in phosphate buffer (pH 7.0) containing 200 mM NaCl. Spectra obtained at 20°C are indicated by the solid line and correspond to the fully folded quadruplex form. Spectra obtained at 95°C are indicated by the dotted line, and correspond to the denatured, unfolded form.

Mentions: The electronic spectroscopies have long been used to characterize the structures of quadruplexes (60), as well as provide a convenient sensitive signal for monitoring transitions or ligand binding. The latter is uncontroversial. CD spectra are routinely used, along with electrophoresis, to assign folds (29,59–65). However, the interpretation of optical properties such as hypochromicity or the shape and sign of CD bands (cf. Figure 5) is controversial (47). Although the CD spectra of A-, B- and Z-DNA are quite different and have been backed by theoretical calculations (66–69), the situation with G-quadruplexes is much less clear. An empirical study that has been much cited showed CD spectra of different structures. However, the authors pointed out that there was no simple relationship between fold and shape of the CD spectrum (47). Although there have been ab initio calculations of the CD of proteins and NAs duplexes (69,70), there has been little work on the theoretical analysis of quadruplex CD. Calculations for an antiparallel DNA d(G4T4)4 (71) showed an essentially conservative CD spectrum in the range 220–320 nm with a maximum at 260 nm and a minimum at 245 nm (zero crossing at 250 nm) that only slightly resembled the experimental spectrum (whose structures were not independently verified). More recently, Gray et al. (72) have carried out calculations for two stacked quartets in which the quartets have the same or opposite polarities for hydrogen bonding (i.e. clockwise or anticlockwise cf. Figure 1). These quartets can stack such that they both have the same polarity or opposite polarity, and specifically the rotation angle between the stacks which gives rise to quite different stacking interactions, which is a major determinant of the intensity and shape of the CD spectrum, and specifically the rotation angle between the stacks. The calculated CD spectra of these two simple states are quite different. The same polarity stacks show a minimum at 235 nm, a maximum at 260 nm (zero crossing at 250 nm) and a second, broad positive band at ∼295 nm (similar to the spectrum often attributed to the parallel conformation). The opposite polarity stacks however gave an inverted spectrum with a quasi conservative spectrum having a minimum at 265 nm, a maximum at 295 nm and a zero crossing at ∼280 nm (often attributed to the antiparallel conformation (cf. Figure 5B). As the authors pointed out, the intensities of the calculated spectra seem to be substantially in error. It is our contention that until either accurate calculations can be done, in which the influence of quartet rotation, additional induced CD from looped bases are systematically accounted for or a rigorous empirical database can be generated, in which the CD spectra of quadruplex samples whose structure has been unequivocally determined on that sample under the same conditions, the interpretation of CD in structural terms is unwise as it amounts to a circular argument.Figure 5.


Stability and kinetics of G-quadruplex structures.

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

Absorbance and CD spectra. UV absorbance (A) and circular dichroic (B) spectra of the human telomere quadruplex sequence 5′AGGG(TTAGGG)3 in phosphate buffer (pH 7.0) containing 200 mM NaCl. Spectra obtained at 20°C are indicated by the solid line and correspond to the fully folded quadruplex form. Spectra obtained at 95°C are indicated by the dotted line, and correspond to the denatured, unfolded form.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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Figure 5: Absorbance and CD spectra. UV absorbance (A) and circular dichroic (B) spectra of the human telomere quadruplex sequence 5′AGGG(TTAGGG)3 in phosphate buffer (pH 7.0) containing 200 mM NaCl. Spectra obtained at 20°C are indicated by the solid line and correspond to the fully folded quadruplex form. Spectra obtained at 95°C are indicated by the dotted line, and correspond to the denatured, unfolded form.
Mentions: The electronic spectroscopies have long been used to characterize the structures of quadruplexes (60), as well as provide a convenient sensitive signal for monitoring transitions or ligand binding. The latter is uncontroversial. CD spectra are routinely used, along with electrophoresis, to assign folds (29,59–65). However, the interpretation of optical properties such as hypochromicity or the shape and sign of CD bands (cf. Figure 5) is controversial (47). Although the CD spectra of A-, B- and Z-DNA are quite different and have been backed by theoretical calculations (66–69), the situation with G-quadruplexes is much less clear. An empirical study that has been much cited showed CD spectra of different structures. However, the authors pointed out that there was no simple relationship between fold and shape of the CD spectrum (47). Although there have been ab initio calculations of the CD of proteins and NAs duplexes (69,70), there has been little work on the theoretical analysis of quadruplex CD. Calculations for an antiparallel DNA d(G4T4)4 (71) showed an essentially conservative CD spectrum in the range 220–320 nm with a maximum at 260 nm and a minimum at 245 nm (zero crossing at 250 nm) that only slightly resembled the experimental spectrum (whose structures were not independently verified). More recently, Gray et al. (72) have carried out calculations for two stacked quartets in which the quartets have the same or opposite polarities for hydrogen bonding (i.e. clockwise or anticlockwise cf. Figure 1). These quartets can stack such that they both have the same polarity or opposite polarity, and specifically the rotation angle between the stacks which gives rise to quite different stacking interactions, which is a major determinant of the intensity and shape of the CD spectrum, and specifically the rotation angle between the stacks. The calculated CD spectra of these two simple states are quite different. The same polarity stacks show a minimum at 235 nm, a maximum at 260 nm (zero crossing at 250 nm) and a second, broad positive band at ∼295 nm (similar to the spectrum often attributed to the parallel conformation). The opposite polarity stacks however gave an inverted spectrum with a quasi conservative spectrum having a minimum at 265 nm, a maximum at 295 nm and a zero crossing at ∼280 nm (often attributed to the antiparallel conformation (cf. Figure 5B). As the authors pointed out, the intensities of the calculated spectra seem to be substantially in error. It is our contention that until either accurate calculations can be done, in which the influence of quartet rotation, additional induced CD from looped bases are systematically accounted for or a rigorous empirical database can be generated, in which the CD spectra of quadruplex samples whose structure has been unequivocally determined on that sample under the same conditions, the interpretation of CD in structural terms is unwise as it amounts to a circular argument.Figure 5.

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