Neuraminidase (NA), one of the major surface glycoproteins of influenza A virus (IAV), is an important diagnostic biomarker and antiviral therapeutic target. Cytolysin A (ClyA) is a nanopore sensor with an internal constriction of 3.3 nm, enabling the detection of protein conformations at the single-molecule level. In this study, a nanopore-based approach is developed for analysis of the enzymatic activity of NA, which facilitates rapid and highly sensitive diagnosis of IAV. Current blockade analysis of the d-glucose/d-galactose-binding protein (GBP) trapped within a type I ClyA-AS (ClyA mutant) nanopore reveals that galactose cleaved from sialyl-galactose by NA of the influenza virus can be detected in real time and at the single-molecule level. Our results show that this nanopore sensor can quantitatively measure the activity of NA with 40–80-fold higher sensitivity than those previously reported. Furthermore, the inhibition of NA is monitored using small-molecule antiviral drugs, such as zanamivir. Taken together, our results reveal that the ClyA protein nanopore can be a valuable platform for the rapid and sensitive point-of-care diagnosis of influenza and for drug screening against the NA target.

Although protein-protein interactions (PPIs) are emerging therapeutic targets for human diseases, development of high-throughput screening (HTS) technologies against PPI targets remains challenging. In this study, we propose a protein complex structure to effectively detect conformational changes of protein resulting from PPI using solid-state nanopore for a novel, widely-applicable drug screening method against various PPI targets. To effectively detect conformational changes resulting from PPI, we designed a fusion protein MLP (MDM2-linker-p53TAD), where p53TAD and MDM2 are connected by a 16 amino acid linker. The globular conformation of MLP exhibited a single-peak translocation event, whereas the dumbbell-like conformation of nutlin-3-bound MLP revealed as a double-peak signal. The proportion of double-peak to single-peak signals increased from 9.3% to 23.0% as nutlin-3 concentration increased. The translocation kinetics of the two different MLP conformations with varied applied voltage were analyzed. Further, the fractional current of the intra-peak of the double-peak signal was analyzed, probing the structure of our designed protein complex. This approach of nanopore sensing may be extendedly employed in screening of PPI inhibitors and protein conformation studies.