DNA synthesis technology is the cornerstone of genomics, synthetic biology, and modern molecular biology, playing a significant role in various emerging fields such as information storage, second-generation sequencing, DNA nanotechnology, and research on oligonucleotide-based drugs. Although chemical synthesis currently dominates DNA synthesis, it still faces many challenges that need to be addressed. As the chain lengthens and side reactions occur, the synthesis efficiency and purity continue to decline, and the error rate increases, making it difficult to synthesize long fragments. Moreover, the entire synthesis process relies on toxic reagents, which contradicts the principles of "green chemistry." In this project, our team will solve the above problems through enzymatic DNA synthesis and attempt to develop a new DNA synthesis technology.

1. Project Description

Enzymatic DNA synthesis, as a promising DNA synthesis technology, has been attracting attention since the 1950s, and the method of template-free DNA synthesis is considered to be the most promising solution for the next generation of artificial DNA synthesis. Among them, Terminal deoxynucleotidyl transferase (TdT) is one of the most hopeful DNA polymerases for de novo DNA synthesis because it can add nucleotides randomly to the initiation strand without a template. Although its function was discovered in the 1960s, with the development and promotion of phosphoramidite synthesis, interest in enzymatic DNA synthesis has significantly declined. In recent years, synthetic biology and new data storage technologies have placed higher demands on the synthesis speed and chain length of DNA chains, and TdT has once again attracted great attention. Extensive research has shown that TdT can bind several deoxynucleotides in 1 second and can extend the synthesized DNA to several thousand bases, far exceeding the synthesis length and speed range of commercial phosphoramidite synthesis technology. However, the natural characteristic of TdT to randomly add deoxynucleotides hinders its development.

Therefore, our team hopes to transform the natural TdT through directed evolution to improve its catalytic activity and capacity, and design and synthesize a modified deoxynucleotide substrate with controllable reactivity to achieve orderly and controllable enzymatic DNA synthesis and develop a new DNA synthesis technology.

2. How Our Project Will Solve This Problem

2.1 Directed Evolution of TdT

TdT was originally isolated from calf thymus and later found in the lymphocytes of birds and some mammals. According to a recent report, the wild-type TdT from Zonotrichia albicollis (ZaTdT) has better catalytic activity and capacity compared to 18 other TdT sources. Therefore, we choose to start with ZaTdT, construct ZaTdT mutants through molecular docking combined with site-directed mutagenesis, and purify the wild-type ZaTdT and its mutant protein mutants in vitro using Escherichia coli BL21(ED3). These proteins will be used for subsequent enzymatic reactions to compare their catalytic activities and capacities.

2.2 Design and Synthesis of Deoxynucleotide Substrates with Reversibly Protected 3' Termini

Based on the characteristic of TdT to randomly add deoxynucleotides to the 3' end of DNA molecules, we will chemically modify the 3' ends of the four natural deoxynucleotide substrates, that is, to add a protecting group that can only be removed under specific wavelengths of light, thereby controlling the orderly extension of DNA chains. After obtaining the modified deoxynucleotide substrates, we will test whether the wild-type ZaTdT and its mutant protein mutants can add them to single-stranded DNA chains to verify whether our idea can be realized.

3. References

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