Similarly spectrophotometric titrations were carried out und
Similarly, spectrophotometric titrations were carried out under conditions identical to those used for the fluorescence titrations. exhibited a predominant Pyrimethamine band at around 493 nm. The titrations with all G-quadruplex DNAs resulted in a hypochromic effect along with a bathochromic shift of the absorption maximum (). For the titration with G-quadruplex , the absorption maximum of gradually decreased, and a red shift (36 nm) could be observed along with an isosbestic point at 522 nm (). On the other hand, the effect of double-stranded DNAs on the absorption band of was much smaller than G-quadruple DNAs. These DNAs caused just slight hypochromicity, suggesting that had much stronger interaction with G-quadruplexes than with duplex DNA form, which was in good agreement with that of fluorescence titration experiments. It is a generally accepted concept that unimportant shift in maximum wavelength of absorption is the most possible outcome of groove binding . Thus, the recorded spectra indicated the binding between and G-quadruplex DNA may be in the mode of groove binding rather than that of intercalation binding. To conduct a quantitative analysis of probe , we then measured the sensitivity of toward different concentrations of G-quadruplexes by means of fluorescence spectra. The limit of detection (LOD) is calculated by the equation: LOD = 3σ/. The σ value represents the standard deviation for multiple measurements (∼15 times) of blank solution. The value is the slope derived from the linear range of the fluorescence titration curve with different G-quadruplex DNAs. To get the slope, the fluorescence intensity at 640 nm is plotted as a concentration of each G-quadruplex DNA (). The values of the LODs were shown in . The linear ranges of the fluorescence titration curves for with most of G-quadruplex DNAs ranged from 0 to 0.8 μM. The corresponding LOD values of for G-quadruplex , , , and were 11.9 nM, 6.4 nM, 7.3 nM, 13.6 nM, and 15.3 nM, respectively (). Clearly, the LODs of for different G-quadruplex DNAs in solution were in the nanomolar range. Taken together, such results suggested that had a satisfactory sensing ability. In addition, among the G-quadruplex structures examined, exhibits the best performance with the lower LODs for parallel G-quadruplexes. The binding stoichiometry of with G-quadruplex DNA was also studied by Job plot experiment (). A 1:1 binding model was well-fitted to the titration date of and , and a 1:2 binding model was observed of and . The fluorescence titration curves were further fitted to an independent-site model and the binding constants were obtained as shown in and . The binding constants () between and G-quadruplexes are in the range of 0.33–14.73 × 10 M. Comparison of the values demonstrated that showed weak interactions with hybrid and anti-parallel G-quadruplexes, while had much stronger interactions with parallel G-quadruplexes, which was consistent with the results of LODs. The structures of G-quadruplex DNA are inherently stabilized by the presence of metal ions which coordinated to O6 atoms of guanines in the quartet . Fluorescence titrations were carried out in different concentrations of K ion (0–100 mM) to study the effect of cationic strength on binding to G-quadruplex DNA . In the absence of any metal ions, obvious fluorescence enhancement of was observed. It is reported that some of the quadruplex forming sequences, for example, , are known to form quadruplexes even in the absence of salt . With increasing of K ion concentration, the value of enhancement in the fluorescence intensity of with was increased and the maximal value occurring near 20 mM (). This may because the K ions preferentially stabilized the G-quadruplex with folding structures. However, above this concentration, the fluorescence enhancement displayed a slight decrease. In the case of higher concentration, K ions occupy the available binding sites on G-quadruplex surface and reduce the possibility for compound to bind exterior of G-quadruplex .