Rational Molecular Design to Improve Digital Polymer Readout in Aerolysin‐Based Nanopore Sequencing

ABSTRACT

Nanopore sequencing is a promising method for decoding synthetic digital polymers, but translocation signals are often complex. This study examines the influence of macromolecular design on the accuracy of nanopore sequencing. Nine phosphoramidite monomers were synthesized and used in automated solid‐phase chemistry to generate a broad library of sequence‐defined poly(phosphodiesters) with varying chain lengths and sequences. The molecular uniformity of the polymers was confirmed by mass spectrometry and ion‐exchange HPLC. Evaluation using aerolysin‐based sensing allowed for the analysis of how key molecular parameters, such as monomer hydrophilicity, size, rigidity, and bulkiness, affect translocation events. This analysis enabled the selection of an optimal binary alphabet for nanopore sequencing, comprising a main‐chain cyclohexane 1,4‐dimethyl spacer for bit‐0 and a benzyl side chain for bit‐1. Using a machine learning approach, distinct polymer lengths and sequences could be identified. These results offer a novel pathway toward achieving low‐loss, repeatable, and reliable sequencing of information‐encoding synthetic polymers, thereby advancing the potential of nanopore technology for digital data storage.