Empowering protein single-molecule sequencing: nanopore technology toward sensing gene sequences

The investigation of proteins at the single-molecule level is urgent to reveal the relationship between their structure and function. Unlike traditional techniques for attaining the overall average effect of group systems, nanopore sensing mode can provide information on the characteristics of proteins at the single-molecule level. Assisting with the intensity, frequency, and period of current changes, nanopore sequencing technology is rapidly advancing due to its merits, including fast readout, high accuracy, low cost, and portability. In particular, the single-molecule nanopore sequencing mode enables in-depth studies of DNA-protein interactions, protein conformation, DNA sequencing, and microbial assay, including genome sequencing of new species. This review summarizes the sensing mechanisms of nanopore sequencing technology in DNA damage, DNA methylation, RNA sequencing, and protein post-translational modifications and unfolding, covering both biological and solid-state nanopores. Due to these significant advantages, nanopore sequencing provides new insights into complex biological processes and enables more precise real-time monitoring of molecular changes. Its applications extend to clinical diagnostics, environmental monitoring, food safety, and forensic analysis. Moreover, the review outlines the present challenges faced by nanopore sequencing patterns, such as the choice of raw reagents and the design of special construction, offering a deep understanding of nanoporous single-molecule sensing toward protein sequence information and structure prediction.