Sanger Sequencing of Xeno‐Nucleic Acid
Abstract
Abstract Xeno‐nucleic acids (XNAs) offer biostable genetic polymers for biotechnology and information technology, but are incompatible with prevailing DNA sequencing methodologies. Here we report a Sanger sequencing approach tailored for XNA, enabling direct readout of XNA strands up to 50 bases. This new method relies on a Bst DNA polymerase mutant, which not only recognizes XNA as a template but also efficiently incorporates dideoxyribonucleoside triphosphate (ddNTP) substrates, facilitating sequencing of XNA by synthesis of complementary DNA. The Bst F710Y mutant exhibits at least two orders of magnitude higher activity in catalyzing ddNTP incorporation. Our method demonstrates accurate sequence determination of diverse XNA strands of distinct backbone chemistries such as TNA and FANA. Lastly, we showcase proof‐of‐concept automated XNA sequencing on a genetic analyzer instrument, by leveraging Bst mutant's ability to stochastically incorporate BigDye‐labeled ddNTP substrates in a single reaction. This work establishes a direct sequencing platform for XNA employing a chain termination strategy, and lays a foundation for future de novo identification of functional XNA and development of XNA‐based data storage system.
Article Details
Authors (13)
Yueyao Wang
Ze Zhang
Department of Polymer Science and Engineering
Lekang Chen
State Key Laboratory of Coordination Chemistry Department of Biomedical Engineering College of Engineering and Applied Sciences Chemistry and Biomedicine Innovation Center (ChemBIC) Jiangsu Key Laboratory of Artificial Functional Materials Nanjing University Nanjing Jiangsu 210023 China
Jing Zhu
Hefei National Research Center for Physical Sciences at the Microscale, CAS Key Laboratory of Strongly-Coupled Quantum Matter Physics, Key Laboratory of Surface and Interface Chemistry and Energy Catalysis of Anhui Higher Education Institutes, Department of Chemical Physics
Shunfu Qin
School of Pharmacy Nanjing University of Chinese Medicine Nanjing Jiangsu 210023 China
Dongying Wei
State Key Laboratory of Coordination Chemistry Department of Biomedical Engineering College of Engineering and Applied Sciences Chemistry and Biomedicine Innovation Center (ChemBIC) Jiangsu Key Laboratory of Artificial Functional Materials Nanjing University Nanjing Jiangsu 210023 China
Jibao Zhang
Nanjing Superyears Gene Technology Co., Ltd. Nanjing Jiangsu 211800 China
Feng Han
State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Chemistry and Biomedicine Innovation Center (ChemBIC), ChemBioMed Interdisciplinary Research Center at Nanjing University
Fulong Wang
Qi Lin
Department of Chemistry
Rui Fan
Xintong Li
Shanghai Key Laboratory of Green Chemistry and Chemical Processes, State Key Laboratory of Petroleum Molecular & Process Engineering, School of Chemistry and Molecular Engineering, East China Normal University, North Zhongshan Rd. 3663, Shanghai 200062, China
Hanyang Yu
State Key Laboratory of Coordination Chemistry, Department of Biomedical Engineering, College of Engineering and Applied Sciences, Chemistry and Biomedicine Innovation Center (ChemBIC), ChemBioMed Interdisciplinary Research Center at Nanjing University