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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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Whole-spectra melting data and the test of the two-state assumption. Thermal denaturation of the human telomere quadruplex sequence 5′AGGG(TTAGGG)3 in a solution containing 0.185 M NaCl is shown as monitored by UV absorbance (A) or CD (B). The corresponding two-wavelength parametric plots to test the two-state assumption (144) are shown in (C and D). The nonlearity of the the data in panels C and D indicate that the denaturation of the quadruplex is not a simple two-state process, and the intermediate states must be included in the reaction mechanism.
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Figure 7: Whole-spectra melting data and the test of the two-state assumption. Thermal denaturation of the human telomere quadruplex sequence 5′AGGG(TTAGGG)3 in a solution containing 0.185 M NaCl is shown as monitored by UV absorbance (A) or CD (B). The corresponding two-wavelength parametric plots to test the two-state assumption (144) are shown in (C and D). The nonlearity of the the data in panels C and D indicate that the denaturation of the quadruplex is not a simple two-state process, and the intermediate states must be included in the reaction mechanism.

Mentions: A second pitfall is the common assumption that denaturation reactions are simple two-state processes, and simply pass from a folded ‘native’ state to an unfolded denatured state without any intermediates. The two-state assumption must be justified by some experimental test. A classical test, first utilized for protein denaturation studies, is to obtain denaturation curves by two (or more) different physical methods (144). If transition curves obtained by the multiple methods are exactly superimposable, that is consistent with a two-state mechanism. More recent tests utilizing multiple wavelength data have appeared. A dual- wavelength parametric test for a two-state denaturation transition monitored by spectroscopy was described (145). In this test, data obtained at two different wavelengths are plotted against one another. For a two-state transition, such a plot should be strictly linear. Deviations from strict linear behavior signal that the denaturation process is not two-state, and likely has intermediate states that are significantly populated. Singular value decomposition (SVD) provides an additional test of the two-state assumption (146,147). With modern diode array spectrophotometers, it is easy to collect entire spectra as a function of temperature, instead of single wavelength data. A set of spectra as a function of temperature defines a 3D surface that is easily converted to a matrix. SVD of the matrix rigorously enumerates the number significant spectral species required to account for the spectral changes. For a two-state transition, there should be only two significant spectral species, corresponding to the folded and unfolded forms. Any number of species greater than two indicates a violation of the two-state assumption, and signals the presence of intermediates. SVD (or a similar multivariate analysis method) has been used to characterize the denaturation of G-quadruplex or other four-stranded structures (148,149). Figure 7 shows examples of whole-spectra UV and CD melting data, and the two-wavelength test of the two-state assumption for the thermal denaturation of the human telomere quadruplex in Na+ solution. For both UV and CD datasets, there are clear deviations from strict linearity, a sure indication that the denaturation reaction is not a simple two-state process, and that intermediate states are populated to a significant degree and must be included in any reaction mechanism. SVD analysis cannot be illustrated in a simple way, but the details of such an analysis are illustrated in refs (59,146,149).Figure 7.


Stability and kinetics of G-quadruplex structures.

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

Whole-spectra melting data and the test of the two-state assumption. Thermal denaturation of the human telomere quadruplex sequence 5′AGGG(TTAGGG)3 in a solution containing 0.185 M NaCl is shown as monitored by UV absorbance (A) or CD (B). The corresponding two-wavelength parametric plots to test the two-state assumption (144) are shown in (C and D). The nonlearity of the the data in panels C and D indicate that the denaturation of the quadruplex is not a simple two-state process, and the intermediate states must be included in the reaction mechanism.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553573&req=5

Figure 7: Whole-spectra melting data and the test of the two-state assumption. Thermal denaturation of the human telomere quadruplex sequence 5′AGGG(TTAGGG)3 in a solution containing 0.185 M NaCl is shown as monitored by UV absorbance (A) or CD (B). The corresponding two-wavelength parametric plots to test the two-state assumption (144) are shown in (C and D). The nonlearity of the the data in panels C and D indicate that the denaturation of the quadruplex is not a simple two-state process, and the intermediate states must be included in the reaction mechanism.
Mentions: A second pitfall is the common assumption that denaturation reactions are simple two-state processes, and simply pass from a folded ‘native’ state to an unfolded denatured state without any intermediates. The two-state assumption must be justified by some experimental test. A classical test, first utilized for protein denaturation studies, is to obtain denaturation curves by two (or more) different physical methods (144). If transition curves obtained by the multiple methods are exactly superimposable, that is consistent with a two-state mechanism. More recent tests utilizing multiple wavelength data have appeared. A dual- wavelength parametric test for a two-state denaturation transition monitored by spectroscopy was described (145). In this test, data obtained at two different wavelengths are plotted against one another. For a two-state transition, such a plot should be strictly linear. Deviations from strict linear behavior signal that the denaturation process is not two-state, and likely has intermediate states that are significantly populated. Singular value decomposition (SVD) provides an additional test of the two-state assumption (146,147). With modern diode array spectrophotometers, it is easy to collect entire spectra as a function of temperature, instead of single wavelength data. A set of spectra as a function of temperature defines a 3D surface that is easily converted to a matrix. SVD of the matrix rigorously enumerates the number significant spectral species required to account for the spectral changes. For a two-state transition, there should be only two significant spectral species, corresponding to the folded and unfolded forms. Any number of species greater than two indicates a violation of the two-state assumption, and signals the presence of intermediates. SVD (or a similar multivariate analysis method) has been used to characterize the denaturation of G-quadruplex or other four-stranded structures (148,149). Figure 7 shows examples of whole-spectra UV and CD melting data, and the two-wavelength test of the two-state assumption for the thermal denaturation of the human telomere quadruplex in Na+ solution. For both UV and CD datasets, there are clear deviations from strict linearity, a sure indication that the denaturation reaction is not a simple two-state process, and that intermediate states are populated to a significant degree and must be included in any reaction mechanism. SVD analysis cannot be illustrated in a simple way, but the details of such an analysis are illustrated in refs (59,146,149).Figure 7.

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