Application of Terminal Deoxynucleotidyl Transferase in Biosensors and Nucleic Acid Synthesis

doi:10.13523/j.cb.2101024

China Biotechnology

2021, Vol. 41

Issue (5): 51-64 DOI: 10.13523/j.cb.2101024

Application of Terminal Deoxynucleotidyl Transferase in Biosensors and Nucleic Acid Synthesis

TANG Meng-tong^1,^2,³,WANG Zhao-guan^1,^2,³,LI Jiao-jiao^1,^2,³,QI Hao^1,^2,^3,^**(

)

1 School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
2 Key Laboratory of Systems Bioengineering of Ministry of Education, Syn Bio Research Platform, Tianjin University, Tianjin 300072, China
3 Collaborative Innovation Center of Chemical Science and Engineering, Tianjin University,Tianjin 300072,China

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Abstract

Terminal deoxynucleotidyl transferase (TdT) is a member of the polymerase X family. Unlike typical DNA polymerases, TdT incorporates nucleotides at the 3'-end of single stranded DNA oligo in a unique template-free manner. Moreover, the high tolerance of TdT to substrates allows it to polymerize modified dNTP. Fluorescence-modified dNTPs, biotin-modified dNTPs and even artificial bases can be used as good substrates. These biochemical properties of TdT make it widely used in the fields of biosensing and nucleic acid synthesis, promote the development of many nucleic acid-based tools and methods, and lay the foundation for the development of enzymatic de novo DNA synthesis technology. The latest progress of cartilage 3D bioprinting and the limitations of current technology are also explained.

Key words： Terminal deoxynucleotide transferase Biosensor Modified nucleotides Nucleic acid synthesis

Received: 19 January 2021 Published: 01 June 2021

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Corresponding Authors: Hao QI E-mail: haoq@tju.edu.cn

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Cite this article:

TANG Meng-tong,WANG Zhao-guan,LI Jiao-jiao,QI Hao. Application of Terminal Deoxynucleotidyl Transferase in Biosensors and Nucleic Acid Synthesis. China Biotechnology, 2021, 41(5): 51-64.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2101024 OR https://manu60.magtech.com.cn/biotech/Y2021/V41/I5/51

Fig.1 Domain of X family DNA polymerase and three-dimensional structure of TdT (a) Domain organization of X-family DNA polymerases (b) Three-dimensional structure of TdT

Table 1 Summary of partial biosensors based on TdT

Fig.2 The composition and principle of TdT-based biosensors Biosensor is composed of recognition element, physical and chemical transducer, signal amplification and signal analysis system. When the recognition element detects the target, TdT polymerization reaction is induced to amplify the signal, and then the biological signal is transformed into electrical signal, optical signal and visual signal output

Fig.3 The principle of transforming TdT-mediated DNA amplification signals into electrical signals (a) Electrical signal output based on DNA-wrapped silver nanoclusters (b) Electrical signal output based on G-quadruplexes combined with hemin (c) Electrical signal is generated by hybridization of MB-polyA with poly-T (d) Electrical signal is blocked by the accumulation of large amounts of ssDNA

Fig.4 The principle of TdT-mediated fluorescence biosensor (a) The principle of TdT-combined CRISPR-Cas12a method for UDG and Dam activity assay (b) The principle of exosomes detection based on TdT-mediated signal amplification (c) The principle of the miRNA-21 detection based on duplex-specific nuclease amplification and TdT-mediated CuNCs

Fig.5 The working principle of the exosome detection system based on TdT amplification

Fig.6 The chemical structure modified nucleotide and the application of TdT in nucleic acid modification field

Fig.7 Schematic illustration of the analysis of DNA damage by single-molecule counting

Fig.8 Two strategies for DNA synthesis by enzymatic methods (a) The principle of de novo DNA synthesis using TdT-dNTP conjugate (b) An enzymatic synthesis strategy for storing information in DNA


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