Rapid and sensitive identification of small molecules, nucleic acids and proteins is useful in a wide variety of sensing applications in clinical diagnostics, drug discovery and environment research. It remains a challenge however for a single sensing method to deal with a large variety of analytes that have significantly different physiochemical properties. One possible strategy would be conversion of diverse analyte sensing information into distinct barcodes that are readable by a single method. Although the 0/1 binary of computers is deeply rooted in the hearts of the people, in fact, most of nature are not binary in nature. Nucleic acids are coded in quaternary, proteins are coded in vigesimal, and non-binary codes tend to have higher coding efficiency. Imitation of Nature, thus, the development of a simple encoding technique with a large coding capacity, a simple preparation procedure and an easy decoding solution is urgent.
Recently, Huang Shuo’s group at the State Key Laboratory of Analytical Chemistry for Life in our department reported a non-binary nucleic acid-encoded strategy for universal nanopore sensing. Briefly, each nucleic acid-encoded probe, which is treated as a molecular barcode, is prepared by self-assembly of synthetic nucleic acid oligomers. Each molecular barcode can be directly read by an MspA nanopore acting as a barcode scanner. To generate the barcode signal, each encoded probe is composed of multiple information nodes. During the translocation of the probe would generate nanopore events in a stepwise manner without nanopore sequencing, similar to reading of a barcode (Figure 1). The constituent elements of information nodes are highly variable, and any phosphoramidite can be employed to build the information node (Figure 2). In the experiment, the author successfully constructed 14 fully distinguishable information nodes. Subsequently, nucleic acid-encoded probes with 2/3/4/5 information nodes were designed, and each were highly identifiable and with high consistency. These results also indicate that the developed 14 distinctive information nodes may be applied to build “n” information nodes to producing in principle, up to 14n distinct molecular barcodes. Compared to the binary coding study that is also based on nanopore reading, the results also indicate that a non-binary barcoding system is advantageous by having a higher coding efficiency, a better scalability and diversity.
Figure 1: The schematic diagram of molecular barcode scanning by nanopore.
Figure 2: Distinguishable library of 14 nucleic acid-encoded probes.
In addition, these barcode probes were adapted to detect different antibody proteins or cancer-related microRNAs, suggesting their immediate application in a wide variety of sensing applications. Different detection modes can be used for different analytes.
This study is the first to propose a strategy for non-binary coding based solely on self-assembly of synthetic nucleic acids and direct nanopore decoding. During the translocation of the probe would generate nanopore events in a stepwise manner without, similar to reading of a barcode. Based on the developed 14 distinctive information nodes，up to 14n distinct molecular barcodes can be produced in principle. By introducing recognition sequences or affinity tags to the probe, the non-binary barcoding system with direct nanopore decoding is useful in simultaneously sensing of multiple targets, including small molecules, nucleic acids and proteins in a multiplex and fast manner. The results also indicate that a non-binary barcoding system is advantageous by having a higher coding efficiency and is expected to promise more analyte-specific sensing by introducing more affinity tags in the information nodes.
The related paper entitled Non-binary Encoded Nucleic Acid Barcodes Directly Readable by a Nanopore has been published on Angewandte Chemie International Edition on March 8, 2021 (DOI：https://doi.org/10.1002/anie.202116482，paper link：https://onlinelibrary.wiley.com/doi/10.1002/anie.202116482). Dr. Shuanghong Yan and M.S. Liying Wang from our department are the co-first authors of this paper. Professor Huang Shuo from our department is the corresponding author of the paper. This project was funded by National Natural Science Foundation of China (Grant No. 31972917, No. 91753108, No. 21675083), Supported by the Fundamental Research Funds for the Central Universities (Grant No.020514380257, No.020514380261), Programs for high-level entrepreneurial and innovative talents introduction of Jiangsu Province (individual and group program), Natural Science Foundation of Jiangsu Province (Grant 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. 5431ZZXM1902), Technology innovation fund program of Nanjing University, China Postdoctoral Science Foundation (Grant No. 2021M691508).