Folding Equilibrium Constants of Telomere G-Quadruplexes in Free State or Associated with Proteins Determined by Isothermal Differential Hybridization

Guanine rich (G-rich) nucleic acids form G-quadruplex structures that are implicated in many biological processes, pharmaceutical applications, and molecular machinery. The folding equilibrium constant (K-F) of the G-quadruplex not only determines its stability and competition against duplex formation in genomic DNA but also defines its recognition by proteins and drugs and technical specifications. The K-F is most conveniently derived from thermal melting analysis that has so far yielded extremely diversified results for the human telomere G-quadruplex. Melting analysis cannot be used for nucleic acids associated with proteins, thus has difficulty to study how protein association affects the folding equilibrium of G-quadruplex structure. In this work, we established an isothermal differential hybridization (IDH) method that is able to determine the K-F of G-quadruplex, either alone or associated with proteins. Using this method, we studied the folding equilibrium of the core sequence G(3)(T(2)AG(3))(3) from vertebrate telomere in K+ and Na+ solutions and how it is affected by proteins associated at its adjacent regions. Our results show that the K-F obtained for the free G-quadruplex is within 1 order of magnitude of most of those obtained by melting analysis and protein binding beside a G-quadruplex can dramatically destabilize the G-quadruplex.