Orientation-Locked DNA Origami for Stable Trapping of Small Proteins in the Nanopore Electro-Osmotic Trap

• Published in: Nano letters Vol. 23; no. 3; pp. 788 - 794
• Main Authors: Wen, Chenyu, Bertosin, Eva, Shi, Xin, Dekker, Cees, Schmid, Sonja
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 08.02.2023
• Subjects: DNA; Letter; Nanopores; Proteins; single-molecule detection; electro-osmotic flow; label-free protein trapping; DNA origami; nanopore electro-osmotic trap (NEOtrap)
• ISSN: 1530-6984; 1530-6992
• DOI: 10.1021/acs.nanolett.2c03569

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.