Terminal Conjugation Enables Nanopore Sequencing of Peptides

Nanopore sequencing of peptides holds great promise for single-molecule proteomics, but robust conjugation strategies to adapt native peptides for motor-enzyme-driven translocation have yet to be developed. Here, we establish terminally directed DNA-peptide conjugation chemistry strategies that expand the applicability of nanopore sequencing beyond synthetic model systems to natural peptides. At the N terminus, omniligase catalyzes rapid and peptide ligation of a DNA handle under mild conditions. At the C terminus, photoredox decarboxylative ligation introduces a bioorthogonal linker that enables CuAAC-mediated DNA attachment that ensures proper stretching and translocation of short peptides through the nanopore. Our study reveals that long peptides can be sequenced with single-end conjugation, while short or neutral peptides require threading tails. Positively charged peptides cannot be translocated under the same electric field but can be sequenced after charge neutralization. The data demonstrate controlled nanopore readouts of peptides that differ widely in length, charge, and sequence. This framework establishes a versatile chemical foundation for adapting natural peptides to nanopore sequencing, advancing single-molecule proteomic analysis.