Single Molecule Ratcheting Motion of Peptides in a Mycobacterium smegmatis Porin A (MspA) Nanopore

Abstract

Diverse functions of proteins, including synthesis, catalysis, and signaling, result from their highly variable amino acid sequences. The technology allowing for direct analysis of protein sequences, however, is still unsatisfactory. Recent developments of nanopore sequencing of DNA or RNA have motivated attempts to realize nanopore sequencing of peptides in a similar manner. The core challenge has been to achieve a controlled ratcheting motion of the target peptide, which is currently restricted to a limited choice of compatible enzymes. By constructing peptide–oligonucleotide conjugates (POCs) and measurements with nanopore-induced phase-shift sequencing (NIPSS), direct observation of the ratcheting motion of peptides has been successfully achieved. The generated events show a clear sequence dependence on the peptide that is being tested. The method is compatible with peptides with either a conjugated N- or C-terminus. The demonstrated results suggest a proof of concept of nanopore sequencing of peptide and can be useful for peptide fingerprinting.

Multiple rereads of single proteins at single–amino acid resolution using nanopores

“…Using a ClpX protein unfoldase to pull a peptide through a nanopore yielded signals that effectively distinguished between different peptides (9), but these reads were difficult to interpret, in part due to the irregular stepping behavior of ClpX (10). Here, we instead applied the precise stepwise control of a DNAtranslocating motor (11)(12)(13) to pull a peptide through a nanopore, similarly to simultaneous work by Yan et al (14) but presenting several key advances: the use of a helicase that pulls the polymer through MspA in smaller, half-nucleotide steps, the ability to identify single amino acid substitutions, and the capability to obtain high-fidelity signals by rereading the same single molecule multiple times. We developed a system in which a DNA-peptide conjugate was pulled through a biological nanopore by a helicase that was walking on the DNA section (Fig.…”

An engineered third electrostatic constriction of aerolysin to manipulate heterogeneously charged peptide transport

“…Given the success of nanopore single-molecule DNA sequencing, 1–3 more studies are focused on single protein sensing and sequencing with biological nanopores. 4–7 Currently, two potential strategies have been proposed for nanopore protein sequencing. One method is to adopt a protein fingerprinting approach like the shotgun proteomics used in mass spectrometry.…”

Controlled movement of ssDNA conjugated peptide through Mycobacterium smegmatis porin A (MspA) nanopore by a helicase motor for peptide sequencing application

“…Notably, there have recently been studies that achieved step-like blocking current by applying a similar strategy to peptides with a high percentage of negatively charged amino acid residues, confirming the importance of stretching the peptide inside the nanopore. 37,38 Potential solutions include engineering MspA-M2 to improve the spatial discrimination of its constriction zone, and stretching the peptide chain by applying electro-osmotic flow or via an enzymatic approach. These improvements could pave the way for our strategy to eventually achieve high-throughput, low-cost single molecule peptide sequencing, with enormous potential in scientific discovery and clinical applications.…”