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bioRxiv · 10.1101/2022.09.09.507286

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

Wen, C.·Bertosin, E.·Shi, X.·Dekker, C.·Schmid, S.
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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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Wen, C., Bertosin, E., Shi, X., Dekker, C., Schmid, S.. 2022-09-12. Orientation-locked DNA origami for stable trapping of small proteins in the NEOtrap. https://doi.org/10.1101/2022.09.09.507286

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