With favorable features such as ultra-high sensitivity, high selectivity, rapid analysis, and low sample consumption, single-molecule technology has been an ideal method to identify molecules at low concentrations. A large repertoire of proteinaceous nanopores have been exploited as powerful single-molecule tools which recognize biomolecular entities, including DNA, RNA, proteins, carbohydrates and metal ions. Biological and chemical strategies such as site-directed mutagenesis and sulfhydryl chemistry have been developed and used to expand the applications of nanopores. The ideal scenario to manipulate the properties of nanopore building blocks has not been achieved.
Organisms encode and perform physiological functions through twenty natural amino acids. However, it is not sufficient to modify protein structures and extend protein functions only by using natural amino acids. Genetic code expansion (GCE) has enabled site-specific incorporation of a constellation of unnatural amino acids (UAAs) into proteins in bacteria, eukaryotic cells, and animals via the discovery and synthetic evolution of orthogonal aminoacyl-tRNA synthetase/tRNACUA pairs, allowing the introduction of modified physiochemical and biological properties. The advent of 2022 Nobel Prize-winning bioorthogonal chemistry have provided unprecedented tools for the visualization and analysis of biomolecules within their native environment. Theemergence of unnatural amino acids bearing bioorthogonal reactive handles has greatly expanded the application scenarios.
Is it possible to develop a clickable biological nanopore to expand its function? Recently, Prof. Ran Xie’s and Prof. Shuo Huang’s group reported the genetically encoded expansion strategy to introduce bioorthogonal reactive sites to the rim of Mycobacterium smegmatis porin A (MspA) nanopore. The wild-type MspA is an octamer. The authors used the orthogonal aminoacyl-tRNA synthetase/tRNACUA pairs to site-specifically introduce the AzK, an unnatural amino acid containing azido group, to position 56 of MspA. Gel electrophoresis, mass spectroscopy and single-channel recording was used to prove the successful preparation of octameric M2 MspA-D56AzK. The AlkK with an alkynyl group was also successfully introduced to MspA, confirming that GCE strategy is highly versatile and is compatible with various types of UAAs during pore engineering.
Figure1. Site-specifically incorporation of unnatural amino acids to MspA nanopore.
To verify the reactivity of the introduced UAAs, DBCO (dibenzocyclooctyne) functionalized single-stranded DNA or lysozyme was covalently coupled to the pore rim via click reaction, allowing real-time observation of the movement of biomacromolecules at single-molecule level. Specifically, by conjugating a lysozyme to the pore rim, the pore immediately gains the capacity of oligosaccharides sensing. This chimeric nanopore demonstrates a clear discrimination between N-acetylated chitotriose (NAG3) and N-acetylated chitohexaose (NAG6). This nanopore-enzyme coupling strategy can monitor the enzymatic motions in real time and provide more dynamic information and measurement continuity.
Figure2. Single-molecule observations of lysozyme-conjugated nanopores bound with different saccharide substrates.
The related paper entitled “Site-Specific Introduction of Bioorthogonal Handles to Nanopores by Genetic Code Expansion” has been published on Angewandte Chemie International Edition on March 16, 2023 (Paper link: https://doi.org/10.1002/anie.202216115,DOI: 10.1002/anie.202216115). Prof. Ran Xie and Prof. Shuo Huang from our department are co-corresponding authors. Ph.D. students Jing Yang and Kefan Wang are co-first authors. This research was supported by the State Key Laboratory of Analytical Chemistry for Life Sciences, and Chemistry and Biomedicine Innovation Center, Nanjing University (ChemBIC), and funded by National Key R&D Program of China (Grant No. 2022YFA1304602), National Natural Science Foundation of China (No. 2207070006, No.22225405, No. 31972917 ), the Fundamental Research Funds for the Central Universities (Grant No.020514380257), Programs for High-level Entrepreneurial and Innovative Talents Introduction of Jiangsu Province (Individual and Group Program), Natural Science Foundation of Jiangsu Province (No. BK20202299, No. BK20200009 ), Excellent Research Program of Nanjing University (Grant No. ZYJH004), Shanghai Municipal Science and Technology Major Project, State Key Laboratory of Analytical Chemistry for Life Science (Grant No. 5431ZZXM2204), China Postdoctoral Science Foundation (Grant No. 2021M691508, Grant No. 2022T150308).