Fig. 1: OPO library preparation and peptide sensing.

a Workflow for OPO library preparation: colored lines represent different peptide fragments featuring a lysine residue at the C-terminus; red dots represent the terminal azido modification; light green anchor integrated with a red dot is to describe the click chemistry linkage; sequencing adaptor is marked as double-stranded blue lines. LysC digestion and azidation modification typically require 0.5 h, though total reaction time may extend to 3 h for hard-to-digest proteins or peptides with limited solubility. b (top) Azido conditions: Peptide and FSO₂N₃ in 1:3 molar ratio; KHCO₃ (100 mM), MTBE/DMF/H₂O (10 μL, 95:2:3, v/v/v), room temperature (RT), 30 min; the incubation may be extended up to 3 h to enhance yield. (bottom) Library design and DNA-templated conjugation (DTC): DBCO modifications present at the 3’-end of DNA1 and the 5’-end of DNA2; Two AP sites, represented as short yellow markers, are placed in both DNA1 and DNA2, and another AP site is in DNA3; Click chemistry reaction was carried out in 1×PBS buffer at RT. c A typical sensing reads from peptide hp1_5 (VERIK): AP site-induced two current peaks (red star) of up to 110 pA enable the algorithmic extraction of OPO signal windows by establishing a threshold at 0.4698 × I₀. d (left) Schematic of nanapore sensing of the OPO libraries. An insulating membrane containing an embedded CsgG nanopore separates cis and trans chambers equipped with electrodes. A sequencing adapter of the OPO library molecule, attached with a T4 Dda helicase motor, is captured by the nanopore. Under optimized ionic conditions (including ATP and MgCl₂), the ATP-dependent helicase motor controls the translocation of the OPO structure through the nanopore; (middle) The linear correlation of the length of uncharged peptides vs. τ_off with R² = 0.9601; (right) The linear correlation of the molar volume of uncharged peptides vs. the value of (1 − I/I₀) × τ_off with R² = 0.9476.