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Orientation-locked DNA origami for stable trapping of small proteins in the NEOtrap

Wen, C.; Bertosin, E.; Shi, X.; Dekker, C.; Schmid, S.

2022-09-12 biophysics
10.1101/2022.09.09.507286 bioRxiv
Show abstract

Nanopores are versatile single-molecule sensors that offer 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 a molecular mass of 14 kDa. The cholesterol functionalization significantly expands the application range of the NEOtrap technology.

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