Biological nanopores crucially control the import and export of biomolecules across lipid membranes in cells. They have found widespread use in biophysics and biotechnology, where their typically narrow, fixed diameters enable selective transport of ions and small molecules, as well as DNA and peptides for sequencing applications. Yet, due to their small channel sizes, they preclude the passage of large macromolecules, e.g., therapeutics. Here, the unique combined properties of DNA origami nanotechnology, machine-inspired design, and synthetic biology are harnessed, to present a structurally reconfigurable DNA origami MechanoPore (MP) that features a lumen that is tuneable in size through molecular triggers. Controllable switching of MPs between 3 stable states is confirmed by 3D-DNA-PAINT super-resolution imaging and through dye-influx assays, after reconstitution of the large MPs in the membrane of liposomes via an inverted-emulsion cDICE technique. Confocal imaging of transmembrane transport shows size-selective behavior with adjustable thresholds. Importantly, the conformational changes are fully reversible, attesting to the robust mechanical switching that overcomes pressure from the surrounding lipid molecules. These MPs advance nanopore technology, offering functional nanostructures that can be tuned on-demand – thereby impacting fields as diverse as drug delivery, biomolecule sorting, and sensing, as well as bottom-up synthetic biology.

Nanopores are versatile single-molecule sensors offering a simple label-free readout with great sensitivity. We recently introduced the nanopore electro-osmotic trap (NEOtrap) which can trap and sense single unmodified proteins for long times. The trapping is achieved by the electro-osmotic flow (EOF) generated from a DNA-origami sphere docked onto the pore, but thermal fluctuations of the origami limited the trapping of small proteins. Here, we use site-specific cholesterol functionalization of the origami sphere to firmly link it to the lipid-coated nanopore. We can lock the origami in either a vertical or horizontal orientation which strongly modulates the EOF. The optimized EOF greatly enhances the trapping capacity, yielding reduced noise, reduced measurement heterogeneity, an increased capture rate, and 100-fold extended observation times. We demonstrate the trapping of a variety of single proteins, including small ones down to 14 kDa. The cholesterol functionalization significantly expands the application range of the NEOtrap technology.