Polypeptide translocation and sequencing
We have engineered many variants of the a-hemolysin protein pore for single molecule detection (stochastic sensing), which is effected by monitoring the modulation of an ionic current passing through a single pore. Stochastic sensing allows the analysis of a wide variety of analytes: metal cations, small organic molecules, nucleic acids, proteins, &c.
The spin-out company from the Bayley laboratory, Oxford Nanopore, has used this technology to sequence single molecules of DNA and RNA (https://www.nanoporetech.com/).
Following the success of Oxford Nanopore's MinION sequencer, we have redirected our efforts towards protein characterization, in particular the detection of post-translational modifications and alternative splicing.
Critical issues include the ability to unfold proteins before or during translocation and the means to translocate polypeptides with a low density of heterogeneously distributed charged side chains.
Rosen, C.B., Rodriguez-Larrea, D. and Bayley, H. Single-molecule site-specific detection of protein phosphorylation. Nature Biotechnology 32, 179-181 (2014). doi:10.1038/nbt.2799
Qing, Y. and Bayley, H. Enzymeless DNA base identification by chemical stepping in a nanopore. J. Am. Chem. Soc. 143, 18181-18187 (2021). doi: 10.1021/jacs.1c07497. PMID: 34669377
Bayley, H. Nanopore sequencing: from imagination to reality. Clinical Chemistry 61, 25-31 (2015). DOI: 10.1373/clinchem.2014.223016
Qing, Y. and Bayley, H. Believe the hype: nanopore proteomics is moving forward. GEN Biotechnology 1, 28-29 (2022) DOI: 10.1089/genbio.2022.29008.qba