Investigating C-kit1 G-Quadruplex Stability using Nanopore

Abstract

In an aqueous solution, certain sequences of single-stranded DNAs can fold into dynamic secondary structures, whose stabilities vary with their surrounding environment contents. In this study, we investigated the C-kit1 promoter - a sequence that plays an important role in several human malignancies. C-kit1 is a guanine-rich sequence that can form stable G-quadruplex structure in the presence of cations. In the presence of G-quadruplex on the promoter region, C-kit1 gene is downregulated, leading to suppression of cancer cell proliferation. Our study aimed to evaluate C-kit1 G-quadruplex structural stability dependence on cations and CX-5461, a small G-quadruplex stabilizing agent that is currently in Phase 1 clinical trial. Conventional methods often utilize fluorescence spectroscopy and circular dichroism to study CX-5461 efficiency on stabilizing G-quadruplex. However, these methods cannot provide real-time molecular dynamic sensing of the structure folding/unfolding behavior. Thus, our study utilized the α-hemolysin nanopore as the main tool for single-molecular analysis of C-kit1 G-quadruplex. Specifically, C-kit1 G-quadruplex formation and stability with and without CX-5461 presence was analyzed using α-hemolysin nanopore, circular dichroism and thermal denaturation. Circular dichroism spectra measurements contained a positive peak at 263nm and negative peak at 240nm, confirming G-quadruplex formation with anti-parallel topology in both 1M KCl and 1M NaCl. Nanopore analysis results showed that C-kit1 G-quadruplex stability is cation-dependent (K+>Na+), resulting in in longer current-blockage events in K+ and significantly decreased event capture rate. Addition of CX-5461 drug further increased C-kit1 G-quadruplex stability, resulting in extended current-blockage events (seconds to minutes long) in nanopore study, and a significantly increased melting temperature.