Spectroscopic study on the binding of a cationic porphyrin to DNA G-quadruplex under different K+ concentrations.
ABSTRACT We have performed systematic spectroscopic titrations to characterize the binding reaction of cationic meso-tetrakis(4-(N-methylpyridiumyl))porphyrin (TMPyP4) with the G-quadruplex (G4) of human telomeric single-strand oligonucleotide d[TAGGG(TTAGGG)3T] (S24), for which special effort was made to examine the TMPyP4-G4 binding stoichiometry, the binding modes, and the conformational conversion of the G4 structure under different potassium ion (K+) concentration. It is found that, in the presence of 0, 10 mM, and 100 mM K+, TMPyP4 forms a complex with the anti-parallel G4 in a TMPyP4-to-G4 molar ratio of 5, 5 and 3, respectively, and the increase of K+ concentration would reduce the binding affinity of TMPyP4 to G4. For the TMPyP4-G4 complex, the end-stacking mode and groove binding mode were presumed mainly by the results of time-resolved fluorescence spectroscopy in the three cases. Most importantly, it is found that TMPyP4 can directly induce the formation of the anti-parallel G4 structure from the single-strand oligonucleotide S24 in the absence of K+, and that it can preferentially induce the conformational conversion of the G4 structure from the hybrid-type to the anti-parallel one in the presence of K+.
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- "These molecules can act as effective anticancer agents by stabilizing the telomeric regions of DNA or by regulating gene expression of oncogenes. Some of these molecules also induce the formation of quadruplex structures in G-rich sequences  . But until recently , the energetics of these interactions has not been well studied. "
ABSTRACT: BACKGROUND: Interaction of putative anticancer agent sanguinarine with two quadruplex forming sequences, human telomeric DNA (H24) and NHE III1 upstream of the P1 promoter of c-myc (Pu27), have been studied to understand the structural basis of the recognition. METHODS: Absorption, fluorescence and circular dichroism have been employed to characterize the association. Energetics of the interaction was studied by isothermal titration and differential scanning calorimetry. TRAP assay was done to assess the inhibitory potential of sanguinarine. RESULTS: Absorption and fluorescence studies show that sanguinarine has high binding affinity of ~10(5) M(-1) for both sequences. Binding stoichiometry is 2:1 for H24 and 3:1 for Pu27. Results suggest stacking interaction between planar sanguinarine moiety and G-quartets. Circular dichroism spectra show that sanguinarine does not cause structural perturbation in the all-parallel Pu27 but causes a structural transition from mixed hybrid to basket form at higher sanguinarine concentration in case of H24. The interaction is characterized by total enthalpy-entropy compensation and high heat capacity values. Differential scanning calorimetry studies suggest that sanguinarine binding increases the melting temperature and also the total enthalpy of transition of both quadruplexes. TRAP results show that sanguinarine effectively blocks telomerase activity in a concentration dependent manner in cell extracts from MDAMB-231 breast cancer cell lines. CONCLUSION: These results suggest that there is a difference in the structural modes of association of sanguinarine to the quadruplexes. GENERAL SIGNIFICANCE: It helps to understand the role of quadruplex structures as a target of small molecule inhibitors of telomerase.Biochimica et Biophysica Acta 04/2013; 1830(8). DOI:10.1016/j.bbagen.2013.03.027 · 4.66 Impact Factor
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ABSTRACT: Circular dichroism and differential scanning calorimetry were used to determine the energetics of the conformational switch of the human telomere quadruplex formed by the sequence d[AGGG(TTAGGG)3] between the sodium basket form and the potassium hybrid form. The energy barrier separating the two conformations was found to be modest, only 1.4-2.4 kcal mol(-1). The kinetics of exchange of bound K+ for Na+ cations and the concomitant conformational switch was assessed by measuring time-dependent changes in the circular dichroism spectrum accompanying the cation exchange reaction. The time course of these changes was found to consist of three distinct kinetic processes: a rapid phase that was complete in less than 5 ms followed by two slower phases with relaxation times of 40-50 s and 600-800 s at 25 degrees C and pH 7.0. We interpret these kinetics in terms of a model in which the bound Na+ cations are rapidly replaced by K+ followed by relatively slow structural rearrangements to generate the final K(+)-bound product(s). Circular dichroism studies showed that addition of the porphyrin TmPyP4 promoted conversion of the basket to the hybrid form. The kinetics of the TmPyP4-induced conformational change were the same as those observed for the cation exchange reaction.The Journal of Physical Chemistry B 04/2009; 113(9):2676-83. DOI:10.1021/jp809578f · 3.38 Impact Factor
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ABSTRACT: G-quadruplexes are higher-order nucleic acids structures formed by G-rich sequences that are stabilized by tetrads of hydrogen-bonded guanine bases. Recently, there has been growing interest in the study of G-quadruplexes because of their possible involvement in many biological processes. Isothermal titration calorimetry (ITC) has been proven to be a useful tool to study the energetic aspects of G-quadruplex interactions. Particularly, ITC has been applied many times to determine the thermodynamic properties of drug-quadruplex interactions to screening among various drugs and to address drug design. In the present review, we will focus on the ITC studies of G-quadruplex structures and their interaction with proteins and drugs and the most significant results will be discussed.International Journal of Molecular Sciences 07/2009; 10(7):2935-57. DOI:10.3390/ijms10072935 · 2.34 Impact Factor