A research group led by Professor Hiroshi Abe of the Graduate School of Science and Assistant Professor Masahito Inagaki of the Graduate School of Engineering at Nagoya University, in collaboration with Professor Natsuhisa Oka of the Graduate School of Engineering at Gifu University, has established a technique that uses silver nanoparticles to cleave DNA at any desired position. The technique utilizes a cleavage reaction in which 3'-phosphorothiolate-modified DNA is cleaved at a specific position by silver ions. They achieved a ligation efficiency 2 to 5 times higher than that of the restriction enzyme method and successfully synthesized genes encoding functional proteins that could be expressed within cells. The technique expanding the possibility of long-chain DNA synthesis has social implications in a wide range of areas, including vaccine development. The findings were published in Nucleic Acids Research issued on June 11.
Genome synthesis is a technology for constructing specific DNA sequences for specific purposes. Unlike gene editing, which modifies existing sequences, genome synthesis allows completely new sequences to be designed, but it was difficult to construct long sequences with existing techniques. In existing methods, PCR-amplified DNAs are cut using restriction enzymes, and the resulting sticky ends are joined together using DNA ligase. However, restriction enzymes can only cut specific positions in the sequence. They also generate short sticky ends, only four bases long. It is believed that longer sticky ends are needed to achieve higher ligation efficiency.
In the present study, the research group focused on a reaction reported in the 1990s, in which 3'-phosphorothiolate-modified DNA is cleaved at a specific position by silver ions. They examined whether this reaction could be applied to the production of sticky ends. The reaction takes advantage of the extremely high affinity between sulfur (S) in the phosphorothiolate linkage and silver (Ag).
First, they designed a primer bearing a 3'-phosphorothiolate linkage at its end and performed PCR to confirm that it can effectively function as a primer for DNA amplification. By applying silver nanoparticles to the amplified product, sticky ends could be produced at any desired position.
When silver ions were used instead of silver nanoparticles, cleavage did proceed, but their non-specific binding to DNA caused precipitation, making them unsuitable for practical use. The group also discovered that the smaller the silver nanoparticles, the higher the DNA cleavage activity, and that coating the particle surface with polyethylene glycol can significantly improve particle stability and dispersion in water. By using silver nanoparticles, they achieved a DNA recovery rate of 98%.
Next, they prepared 10-base and 18-base sticky ends and compared ligation efficiency. While the ligation efficiency of the restriction enzyme method (4-base sticky ends) was 8%, the efficiency improved to 39% and 44% by using the 10-base and 18-base sticky ends, respectively. Furthermore, a gene encoding a functional protein (GFP; green fluorescent protein) was synthesized to verify the functionality of the ligated product. They synthesized a promoter sequence and a GFP gene of approximately 830 bp and 1,260 bp, respectively, and used silver nanoparticles to generate 10-base or 18-base sticky ends. These ends were then ligated.
They examined the GFP expression in human cells using fluorescence as an indicator and revealed that the silver nanoparticle method achieved a ligation efficiency more than five times higher than that of the restriction enzyme method.
The method they developed is highly practical. It is less expensive than the restriction enzyme method and, as it is based on chemical reaction, it works efficiently under mild conditions.
Abe stated, "Our laboratory is also developing mRNA vaccines, and we are planning to apply this technique to develop a benchtop platform capable of producing mRNA vaccines at low costs with high precision. Since the technique allows us to design any sequence we want, it should find its application in a wide range of fields, including antibodies, useful substances and synthetic organisms. We would like to expand our work into these areas as well."
Journal Information
Publication: Nucleic Acids Research
Title: Silver nanoparticle-induced site-specific strand cleavage of chemically modified oligonucleotides for long-chain DNA assembly
DOI: 10.1093/nar/gkag525
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