BPS2026 – Measuring nanopore force generation on DNA using single-molecule biotin-streptavidin bond rupture kinetics

Nanopores have emerged not only as powerful tools for sequencing nucleic acids but also for interrogating single-molecule biophysical interactions with unprecedented precision. As novel nanopore techniques continue to develop, there is increasing need to understand the forces which act on biopolymers within the nanopore. Here, we explore the mechanics of force generation in nanopores using the rupture kinetics of single biotin-streptavidin bonds as a point of calibration. In these experiments, streptavidin is bound to a single-stranded DNA (ssDNA) biotinylated at the 3' end. The 5' end of the ssDNA is electrophoretically drawn through a single MspA nanopore until streptavidin comes to rest on the pore rim. After some time, the biotin-streptavidin bond ruptures stochastically. The time from capture to bond rupture is related to the force exerted on the ssDNA, which can be modulated either by changing the applied voltage or by altering the effective ssDNA charge density using a mixture of regular nucleotides and neutrally charged methyl-phosphonates. These measurements, combined with kinetic models of biotin-streptavidin rupture and previous rupture experiments, provide new insights into the mechanism of force generation in nanopore systems and their application to non-uniformly charged polymers.