biostudies-literaturePokhrel PNCI NIH HHSNational Institutes of HealthNational Science FoundationJapan Society for the Promotion of Science114693https://www.ebi.ac.uk/biostudies/studies/S-EPMC9133229biostudies-literatureUnknown649Binding between a ligand and a receptor is a fundamental step in many natural or synthetic processes. In biosensing, a tight binding with a small dissociation constant (K_d) between the probe and analyte can lead to superior specificity and sensitivity. Owing to their capability of evaluating competitors, displacement assays have been used to estimate K_d at the ensemble average level. At the more sensitive single-molecule level, displacement assays are yet to be established. Here, we developed a single-molecule displacement assay (smDA) in an optical tweezers instrument and used this innovation to evaluate the binding of the L2H2-6OTD ligands to human telomeric DNA G-quadruplexes. After measuring K_d of linear and dendrimer L2H2-6OTD ligands, we found that dendrimer ligands have enhanced binding affinity to the G-quadruplexes due to their polyvalent geometry. This increased binding affinity enhanced inhibition of telomerase elongation on a telomere template in a Telomerase Repeated Amplification Protocol (TRAP). Our experiments demonstrate that the smDA approach can efficiently evaluate binding processes in chemical and biological processes.Analytical biochemistrySingle-molecule displacement assay reveals strong binding of polyvalent dendrimer ligands to telomeric G-quadruplex.PMC9133229JP 20J1381420H02876R01 CA236350CBET1904921Pokhrel PKarna DHu CSasaki SMa YMao HPandey SNagasawa KfalseSingle-molecule displacement assay reveals strong binding of polyvalent dendrimer ligands to telomeric G-quadruplex.Binding between a ligand and a receptor is a fundamental step in many natural or synthetic processes. In biosensing, a tight binding with a small dissociation constant (K_d) between the probe and analyte can lead to superior specificity and sensitivity. Owing to their capability of evaluating competitors, displacement assays have been used to estimate K_d at the ensemble average level. At the more sensitive single-molecule level, displacement assays are yet to be established. Here, we developed a single-molecule displacement assay (smDA) in an optical tweezers instrument and used this innovation to evaluate the binding of the L2H2-6OTD ligands to human telomeric DNA G-quadruplexes. After measuring K_d of linear and dendrimer L2H2-6OTD ligands, we found that dendrimer ligands have enhanced binding affinity to the G-quadruplexes due to their polyvalent geometry. This increased binding affinity enhanced inhibition of telomerase elongation on a telomere template in a Telomerase Repeated Amplification Protocol (TRAP). Our experiments demonstrate that the smDA approach can efficiently evaluate binding processes in chemical and biological processes.2022-01-01T00:00:00Z2022 Jul2024-11-12T21:07:40.219Z2024-11-12T21:07:40.219ZS-EPMC91332293550065710.1016/j.ab.2022.114693