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

Area of Science:

• Polymer Chemistry
• Nanotechnology
• Biotechnology

Background:

• Nanopore sequencing offers potential for decoding synthetic digital polymers.
• Translocation signals in nanopore sequencing are often complex and difficult to interpret.
• Macromolecular design significantly influences the accuracy of nanopore sequencing.

Purpose of the Study:

• To investigate how macromolecular design impacts the accuracy of nanopore sequencing.
• To identify optimal molecular parameters for reliable polymer sequencing.
• To develop a method for decoding sequence-defined synthetic polymers using nanopore technology.

Main Methods:

• Synthesized nine phosphoramidite monomers for automated solid-phase chemistry.
• Generated a library of sequence-defined poly(phosphodiesters) with varied lengths and sequences.
• Utilized aerolysin-based nanopore sensing and machine learning for signal analysis.

Main Results:

• Identified key molecular parameters (hydrophilicity, size, rigidity, bulkiness) affecting translocation events.
• Selected an optimal binary alphabet for nanopore sequencing: cyclohexane 1,4-dimethyl spacer (bit-0) and benzyl side chain (bit-1).
• Achieved distinct identification of polymer lengths and sequences using machine learning.

Conclusions:

• Optimized macromolecular design enhances nanopore sequencing accuracy for synthetic polymers.
• This work provides a pathway for low-loss, repeatable, and reliable sequencing of information-encoding polymers.
• Advances nanopore technology for synthetic polymer analysis and digital data storage.