Lately various pathways of human telomere (ht) DNA folding into G-quadruplexes and of ligand binding to these structures have been proposed. the above, we believe that our work Nitisinone sets the framework for better understanding the heterogeneity of ht-DNA folding and binding pathways, and its structural polymorphism. Introduction Guanine-rich DNA sequences in the presence of cations can fold into four-stranded structures called G-quadruplexes. The existence of potential quadruplex sequences in key regions of the eukaryotic genome, including the immunoglobulin heavy chain switch region, promoter regions, ribosomal DNA, oncogenes, and telomeres, suggests that they may play Tm6sf1 an important role in the mechanism and control of several cellular processes (1C3). Therefore, G-quadruplexes are relevant targets of small molecules that can potentially modulate their biological functions, gene expression, and protein synthesis (4,5). Quadruplex topologies may differ in glycosidic bond angles, strand orientation, connecting loop regions, and molecularity leading to conformational heterogeneity of G-quadruplex structures. This is well exemplified by guanine-rich human telomeric (ht) repeat sequences, which are capable of adopting multiple topologies. For example, monomeric ht quadruplexes containing the core sequence d(AGGG(TTAGGG)3) (Tel22) can adopt several distinct quadruplex topologies. X-ray crystallography reveals that in the presence of K+ ions, Tel22 shows all-parallel strand orientation (6) while in K+ solutions it adopts, according to NMR and other biophysical techniques, a (3+1) hybrid-type topology (denoted as 10? 10 stacking interactions (25C28). The analysis of calorimetric (DSC and ITC) and spectroscopic (CD and FL) data obtained in solutions with K+ (see Fig.?2; Figs. S4 and S6) or Na+ ions (Figs. S3, S5, and S7) suggests that the observed unfolding and binding processes may be described by the model mechanism that involves five macroscopic states (Fig.?1). Reversibility of folding/unfolding of Tel22 in the absence of ligands and in the presence of K+ or Na+ ions (? ? ? ? represents the property of the solute at a given pressure, and it includes the temperature and refer to each step in the model mechanism presented in Fig.?1. From Eq. 2 are derived various model functions (see Eqs. S3CS6 and S9 in the Supporting Material) expressed in terms of a set of adjustable parameters that describe the CD (and and through the?Gibbs-Helmholtz relation and the Kirchhoffs law and (note that is given by represents the number of ions released or uptaken in the transition step and is assumed to be independent of (29). Note that equilibrium molar concentration of unbound for each step in the suggested mechanism) define each equilibrium constant, ? equilibrium) in the presence of K+ (Fig.?2, and and ? and ? (24). In K+ or Na+ solutions, can be considered to be a mixture of so-called G-triplex conformations ( ?400 cal mol?1 K?1]. These thermodynamic parameters Nitisinone are comparable with those reported for the thrombin binding aptamer folding/unfolding transition (34). is more thermodynamically stable (is lower) in solutions with K+ than with Na+ ions, which is a general characteristic of the G-quadruplex stability (35,36). Figure 3 Structural features monitored by CD spectroscopy. Spectra corresponding to hybrid (? ? is taken into account, supports the suggested linkage between the folding and binding processes (Figs. 1, ?,2,2, and and ? ? ? is not populated, as well as the model that considers the ? ? equilibrium and assumes that binds to two comparable 3rd party binding sites on (Model 3). As demonstrated in Fig.?S8, Model?2 cannot describe the DSC ITC and thermograms data measured at 35C, while Model 3 does not describe Compact disc titration data. Alternatively, more-complex choices involve way too many adjustable guidelines that are correlated and therefore can’t be determined with adequate accuracy highly. Our analysis stresses an important benefit of the global installing Nitisinone over the original installing from the model to limited datasets (29). For instance, ITC data only (measured at the moment in the perfect solution is), however, relating to other obtainable experimental data (DSC and Compact disc titration), such evaluation leads to thermodynamic binding guidelines which have no physical meaning. Thermodynamics and structural features Compact disc spectra (Figs. 3 and S9) recommend for both ligands (Phen-DC3, 360A-Br) that their binding can be followed by quadruplex conformational adjustments which the ensuing complexes (and ? and ? binding affinity for the 1st ligand molecule can be greater than for the next ligand (Desk S2). Both measures are enthalpy-driven, followed by negative modification in entropy and temperature capability (Fig.?4 and.