Prof. Tingjian Chen’s Team Publishes a Paper in Nucleic Acids Research: Recognition of Unnatural Base Pairs by a Eukaryotic DNA Polymerase and Development of a Universal Sequencing Technology for an Expanded Genetic Alphabet

Recently, the research team led by Professor Tingjian Chen from the School of Biology and Biological Engineering at South China University of Technology has made significant progress in exploring the recognition of unnatural base pairs (UBPs) by a eukaryotic DNA polymerase and its application in developing DNA sequencing technology for an expanded genetic alphabet. The team systematically investigated and demonstrated for the first time the ability of human DNA polymerase β (Pol β) to recognize a panel of representative UBPs. Based on this discovery, they creatively developed a universal and precise sequencing method for DNA containing unnatural bases, successfully reading sequences with single or multiple unnatural bases. This achievement has been published in the top-tier international journal Nucleic Acids Research, entitled “Recognition of unnatural base pairs by a eukaryotic DNA polymerase enables universal sequencing of an expanded genetic alphabet”. Master's student Hantao Luo, Associate Researcher Yuhui Du, and Postdoctoral Fellow Leping Sun are co-first authors of the paper. Professor Tingjian Chen is the sole corresponding author, and South China University of Technology is the sole completing institution.  (Link to the paper: https://doi.org/10.1093/nar/gkaf1460)

The genetic information of living organisms is encoded by four natural bases: A, T, C, and G. To overcome the coding limits of natural genetics, scientists aim to introduce replicable UBPs into DNA, constructing an expanded genetic alphabet. However, two major challenges are whether these UBPs can be effectively recognized and replicated by the eukaryotic DNA replication machinery, and how to efficiently sequence DNA containing unnatural bases.

In this work, the team first explored and systematically characterized the ability of Pol β to recognize, synthesize, and extend past a panel of representative unnatural base pairs and their derivatives (including dNaM-dTPT3, dNaM-dTPT3Al, dNaM-dTPT3Am, dCNMO-dTPT3, dCNMO-dTPT3Al, and dCNMO-dTPT3Am) during primer extension, DNA gap filling, and subsequent strand-displacement synthesis. The results showed that Pol β could efficiently and accurately synthesize various representative UBPs, including dNaM-dTPT3 and dCNMO-dTPT3, during primer extension or gap filling, and further extend the primer or perform strand-displacement synthesis afterward, demonstrating excellent catalytic efficiency and substrate specificity. Subsequently, leveraging Pol β’s strict discrimination between natural and unnatural substrates, the team innovatively developed a novel universal sequencing strategy for DNA containing unnatural bases. The core steps of this method include: 1) Using Pol β for primer extension, which stalls precisely before the unnatural base site; 2) Employing Taq DNA polymerase to selectively convert the unnatural base into two different natural bases (e.g., dA or dC) in parallel reactions, followed by further primer extension; 3) Identifying the exact position of the unnatural base by comparing the peak area ratios of different natural bases at the corresponding position in the conventional Sanger or deep sequencing results from the two parallel reaction products. This method was successfully applied to sequence fixed sequences containing single or multiple unnatural bases, as well as a random-sequence library containing a single unnatural base, demonstrating excellent accuracy and universality.

This study provides the first molecular-level evidence that the eukaryotic DNA replication and repair systems possess the potential to recognize and process UBPs. It paves the way for future expansion of the genetic alphabet and genetic code in yeast, plant, animal cells, and even more complex eukaryotic systems, towards the construction of eukaryotic semi-synthetic organisms (eSSOs). The established sequencing technology for unnatural base-containing DNA is simple to operate, yields precise results, is applicable to various DNAs containing different unnatural bases, and is directly compatible with mainstream Sanger sequencing and high-throughput sequencing platforms. It offers a stable, reliable, and easily adoptable sequencing solution for DNA fragments with an expanded genetic alphabet, laying a crucial technical foundation for the practical application of unnatural bases in fields such as DNA data storage, molecular evolution (e.g., SELEX), and biomedical diagnostics. This work holds significant scientific importance and broad application prospects. 

This work was supported by the National Natural Science Foundation of China, Program for Guangdong Introducing Innovative and Entrepreneurial Teams, Guangdong Provincial Pearl River Talents Program, National Key R&D Program of China, and Science and Technology Project of Guangzhou, China.