Chinappi, M. | bioRxiv Science

bioRxiv2026

β-Barrel domain swapping in α-hemolysin enables enhanced single-molecule biomolecule sensing

Biological nanopores are powerful platforms for single-molecule analysis, yet rational strategies to tune their transport and sensing properties remain limited. Here we present a modular engineering approach based on {beta}-barrel domain swapping to reprogram the function of the prototypical nanopore -hemolysin. By replacing its native {beta}-barrel with {beta}-barrel domains from diverse pore-forming toxins, we generate a series of chimera nanopores that retain oligomerization capability while exhibiting reduced membrane-permeabilizing activity. Electrophysiological measurements show that selected chimera pores form stable, conductive channels with distinct ion transport properties. Notably, the HL_NetB chimera displays stable conductance, enhanced electroosmotic flow, and improved performance in nucleic acid and protein sensing. Single-molecule experiments demonstrate that this chimera markedly slows the translocation of single-stranded DNA, enabling discrimination by length and sequence, improves the resolution of intrinsically disordered proteins such as -synuclein, and enhances sensitivity to RNA conformational changes. Together, these results establish {beta}-barrel domain swapping as a general and effective strategy for engineering biological nanopores with tailored single-molecule sensing capabilities.

biochemistry↗

bioRxiv2023

An engineered electroosmotic flow transports unravelled proteins across nanopores

The development of a technology capable of sequencing single proteins holds promise to unravel new biological information hidden in ensemble analysis. However, new techniques must be first developed. In one approach, proteins are unfolded and translocate across a nanopore under an external bias. Unlike DNA, however, proteins do not have a uniform charge, and the electrophoretic force cannot be used to translocate proteins. Here, we show that by introducing sets of charges spaced by ~1 nm an otherwise neutral nanopore an electroosmotic force is created that induces the unidirectional transport of polypeptides, even against relatively strong electrophoretic forces. Unstructured polypeptides and native proteins unfolded with urea produce current signatures as they traversed the nanopore, which could lead to quick protein identifcation. This approach can be used to translocate and stretch proteins in non-enzymatic protein identification and enzymatic protein sequencing approaches.

biophysics↗