Differential Antisense Hybridization to Screen G-Quadruplex Stabilizers by Using Isothermal Titration Calorimeter

This work intended to establish a novel method to screen G-quadruplex ligands that stabilize the G-quadruplex structures formed by Hoogsteen Hydrogen bond of guanine-rich sequences. Telomere, in the end of chromosomes, has guanine-rich sequences, these sequences are prone to adopt G-quadruplexes in vivo. These unusual structures might play an important role in biological processes, especially inhibiting the elongation of telomerase, an enzyme essential for immortalization of tumor cell. Thus, G-quadruplexes are likely as the promising target for anticancer drug design. Since a number of small molecules were reported, a novel method is requested to examine if they really have the ability to stabilize G-quadruplex structure. In general, a thermal melting analysis is a popular method, in which the unfolding of G-quadruplex is monitored by changes in spectroscopic signal. One can screen the G-quadruplex stabilizer by comparing melting temperature (Tm) before and after adding small molecules to G-quadruplexes. However, Tm of human telomeres is normally much higher than the physiological temperature. At high temperature, some compounds would induce structural change of G-quadruplexes in 150 mM K+ buffer condition, but this conversion would not happen at the physiological temperature. Moreover, melting analysis cannot be applied to G-quadruplexes associated with the related proteins because of the tolerance of the proteins.
In the work, we established an isothermal method to screen G-quadruplex stabilizers based on antisense hybridization. We used isothermal titration calorimetry (ITC) to investigate the effects of each G-quadruplex ligand on duplex formation by dAG3(T2AG3)3 and its complementary strand d (C3TA2)3C3T. It is believed the better the G-quadruplex stabilizer, the less the duplex formation. Therefore, the binding stoichiometry ratio (N) for the duplex formation is small. Accordingly, we have successfully distinguished those G-quadruplex stabilizers in 150 mM K+ buffer condition, in which those stabilizers could not be distinguished by CD melting analysis. We found that the order of stabilizing ability is 8C3O > Braco19 > BMVC-4 > BMVC > BMVC-5 > TMPyP4. In addition, we can combine equilibrium constant of hybridization from this method and Hess’s law to derive the stabilizer selectivity to G-quadruplex from duplex. The results are in agreement with those K ratio obtained by fluorescent titration. Among these stabilizers, 8C3O has best selectivity owing to its hydrophobic property. In summary, we developed a mimic physiological method to screen G-quadruplex stabilizers. We believe that this method can provide more reliable information for drug design.