Single-molecule investigation of human telomeric G-quadruplex interactions with Thioflavin T

Abstract G-quadruplex ligands have been accepted as potential therapeutic agents for anticancer treatment. Thioflavin T (ThT), a highly selective G-quadruplex ligand, can bind G-quadruplex with a fluorescent light-up signal change and high specificity against DNA duplex. However, there are still different opinions that ThT induces/stabilizes G-quadruplex foldings/topologies in human telomere sequence. Here, a sensitive single-molecule nanopore technology was utilized to analyze the interactions between human telomeric DNA (Tel DNA) and ThT. Both translocation time and current blockade were measured to investigate the translocation behaviors. Furthermore, the effects of metal ion (K + and Na + ) and pH on the translocation behaviors were studied. Based on the single-molecule level analysis, there are some conclusions: (1) In the electrolyte solution containing 50 mmol/L KCl and 450 mmol/L NaCl, ThT can bind strongly with Tel DNA but nearly does not change the G-quadruplex form; (2) In the presence of high concentration K + , the ThT binding induces the structural change of hybrid G-quadruplex into antiparallel topology with an enhanced structural stability; (3) In either alkaline or acidic buffer, G-quadruplex form will change in certain degree. All above conclusions are helpful to deeply understand the interaction behaviors between Tel DNA and ThT. This nanopore platform, investigating G-quadruplex ligands at the single-molecule level, has great potential for the design of new drugs and sensors.