Nanopore sequencing with proteins: synchronization and dischronization of molecular dynamics simulations with laboratory and industrial developments.

Abstract

Protein nanopores have revolutionized DNA sequencing by enabling long-read, real-time, and portable genomic analysis. This review traces the experimental evolution of three key protein nanopores, namely, α-hemolysin, MspA, and CsgG, highlighting how iterative engineering overcame challenges such as translocation control and homopolymer resolution. Concurrently, molecular dynamics (MD) simulations have elucidated DNA-pore interactions, ionic current modulation, free-energy landscapes, and so forth, providing mechanistic insights and guiding rational design. However, MD studies consistently lag behind experimental and industrial advances, resulting in a reactive "simulate-after-validate" paradigm. We identify critical gaps in simulating motor-pore complexes, experimental timescales, and emerging designs, like dual-constriction pores. To bridge these, we propose leveraging deep learning-based structure prediction, <i>de novo</i> protein design, and advanced multiscale simulations to foster the proactive, integrated development of next-generation nanopore technologies.