Angew. Chem.：Unnatural amino acids facilitate nanopore peptide sequencing

One of the key issues in nanopore protein sequencing technique is how to improve the spatial resolution of nanopore sensing interfaces that are dedicatedly designed and gifted by nature. Recently, the group of Prof. Yi-Tao Long of Nanjing University and Prof. Peng R. Chen of Peking University have applied the pyrrolysine-based genetic code extension technique to the Aerolysin nanopore interface through a long-term cooperation and exploration. During a 4-year study, significant progress was made in achieving a highly efficient incorporation of unnatural amino acids into the sensing region of a nanopore. This breakthrough allowed for the precise design and regulation of the spatial orientation of each amino acid within the sensitive region. As a result, a single-molecule interface with a controlled three-dimensional chemical and physical environment was successfully obtained (Figure 1).

Figure 1. Nanopore sensitive region constructed from unnatural amino acids for single peptide sensing

Both research groups dedicated their efforts to increasing the yield of unnatural amino acid-incorporated pore-forming proteins to 5 mg/L. This was achieved through the evolution of efficient aminoacyl-tRNA synthetase and optimization of the expression conditions for the Aerolysin protein (Figure 2). Remarkably, the standard deviation of open-pore current observed in the experiments involving the unnatural amino acid-incorporated proteins was comparable to that of the wild-type proteins. This finding provides strong evidence for the high homogeneity of the products generated using the genetic code extension technique.

Figure 2.High expression system of Aerolysin pore-forming protein encoded by unnatural amino acids.

The sensing region, constructed using unnatural amino acids, was further performed analysis through molecular dynamics simulations and single-molecule experiments. A comparison was made between the behavior of natural amino acids and longer unnatural aromatic amino acid side chains within the engineered nanopore. It was observed that the longer aromatic side chains inside the UAA functionalized nanopore exhibited a favorable geometric orientation for single peptide sensing, contrasting with the wall-adhering behavior of natural amino acids inside the K238F Aerolysin. As a result, the duration time of peptide was prolonged by the UAA functionalized Aerolysin, achieving the discrimination of peptides of different lengths in the mixture (Figure 3).

Figure 3. All-atom model of aromatic nanopore and results of peptide single-molecule sensing.

This method breaks through the limitations of conventional methods for protein nanopore modification, which establishes a high expression system for pore-forming proteins encoded by unnatural amino acids. This study provides a new framework to endow unique sensing properties to nanopore for single peptide sequencing.

This research has been published online in Angew. Chem. Int. Ed. entitled “Controlled Genetic Encoding of Unnatural Amino Acid in Protein Nanopore” (Paper link: https://doi.org/10.1002/anie.202300582，DOI: 10.1002/anie.202300582). The postdoctoral fellow Dr. Xue-Yuan Wu from our department is the first author of the paper, Prof. Yi-Lun Ying and Prof. Chen Peng from Peking University are the co-corresponding authors. This research was supported by the National Key R&D Program of China (2022YFA1304604), National Natural Science Foundation of China (22027806, and 22090050), and the Postdoctoral Science Foundation of China (2021M691509).