Regulatory-sequence mechanical biosensor: A versatile platform for investigation of G-quadruplex/label-free protein interactions and tunable protein detection

Highlights: • Biomolecular force measurement and tunable detection of proteins have been integrated into one mechanical biosensor. • The binding force of DNA G-quadruplexes and label-free protein can be determined by Regulatory-sequence force spectroscopy. • The dynamic range for detecting thrombin concentrations was over eight orders of magnitude. • The influence of a ligand on the interaction force of telomeric GQ/Ku protein complex has been explored. -- Abstract: Mechanical biosensors can be used to quantitatively explore DNA-protein binding mechanisms by detecting targets at low concentrations or measuring force in single-molecule force spectroscopy. However, restrictions in single-molecule manipulation and labelling protocols have hindered the application for bulk analysis of label-free protein detection. Here, we present the integration of molecular force measurement and finely tunable detection of label-free proteins into one mechanical sensor. Regulatory-sequence force spectroscopy was obtained to investigate the binding force of DNA G-quadruplexes (GQ) and label-free protein. The dual control of regulatory sequences and mechanical forces induces the structure switching from DNA duplex to GQ/protein complex. It exhibits a synergistic effect, enabling the rational fine-tuning of the dynamic range for biosensing protein concentrations over eight orders of magnitude. Furthermore, this method was exploited to estimate the stability of the human telomeric DNA GQ by Ku protein and ligand methylpyridostatin. The results revealed that human telomeric GQ has two different binding sites for Ku protein and ligand. Force spectroscopy integrating label-free force measurement and tunable target detection holds great promise for use in biosensing, drug screening, targeted therapies, DNA nanotechnology, and fields in which GQ are of rapidly increasing importance.