Multi-pass nanopore for single-molecule protein sequencing

Protein sequencing using nanopores comes with many challenges. 

In this new method, the authors use the ClpX unfoldase to ratchet proteins through a nanopore. This multi-pass nanopore for single-molecule protein sequencing allowed rereading the same peptide strand multiple times providing higher resolution readouts.

The authors used a two-step process. First, they attached a negatively charged ‘tail’ to the protein of interest, so that the resulting molecule could be rapidly threaded through a nanopore using an electric field. A bulky ‘stopper’ was also attached, to prevent the threaded protein from passing completely through the nanopore. The authors then used an enzyme called ClpX (which unfolds proteins in cells as part of a protein-degradation process) to pull the molecule back through the pore in a way that sequentially presents parts of the threaded protein to the nanopore constriction. 

https://www.nature.com/articles/s41586-024-07935-7

https://sciencemission.com/Multi-pass,-single-molecule-nanopore-reading-of-long-protein-strands