Contribution of potassium ion and split modes of G-quadruplex to the sensitivity and selectivity of label-free sensor toward DNA detection using fluorescence

In recent years, bioanalytical technology based on G-quadruplex has been paid significant attention due to its versatility and stimulus-responsive reconfiguration. Notwithstanding, several key issues for template-directed reassembly of G-quadruplex have not been resolved: what is the key factor for determining the sensitivity and selectivity of split G-quadruplex probes toward target DNA. Therefore, in this study, we designed three pairs of split G-quadruplex probes and investigated the sensitivity and selectivity of these systems in terms of potassium ion concentration and split modes of G-quadruplex. Due to its simplicity and sensitivity, N-methyl-mesoporphyrin (NMM) as fluorescence probes was used to monitor the target-directed reassembling process of G-quadruplex. A G-quadruplex sequence derived from the c-Myc promoter was split into "symmetric" probes, where each fragment contained two runs of guanine residues (2+2), or into "asymmetric" fragments each containing (3+1 or 1+3) runs of guanine residues. In all three cases, the sensitivity of target detection was highly dependent on the thermodynamic stability of the hybrid structure, which can be modulated by potassium ion concentrations. Using a combination of CD, fluorescence, and UV spectroscopy, we found that increasing potassium concentrations can increase the sensitivity of target detection, but can decrease the selectivity of discriminating cognate versus mismatched "target" DNA. The previous argument that asymmetrically split probes were always better than symmetrically split probes in terms of selectivity was not plausible anymore. These results demonstrate how the sensitivities and selectivity of split probes to mutations can be optimized by tuning the thermodynamic stability of the three-way junction complex.