DNA is composed of long chains that act as the blueprint for living organisms. In genetic engineering, scientists cut DNA at specific sites and join the resulting fragments to other DNA sequences, enabling applications such as advanced crop breeding, genetic disease treatment, and the generation of animal models for drug discovery.
Assembling short DNA fragments requires overhanging sequences, known as sticky ends, to facilitate efficient binding. However, generating sticky ends requires precise cutting at targeted sites, which remains challenging with current technologies.
A Japanese research group has developed a silver nanoparticle-based technology to precisely cut and join DNA at targeted sites, achieving two to five times higher DNA assembly efficiency than conventional restriction enzyme methods. These findings were published in the journal Nucleic Acids Research.
Traditional long-chain DNA assembly uses restriction enzymes to cut DNA and T4 DNA ligase to reconnect the fragments. However, restriction enzymes cut only at specific sequences and generate sticky ends that are often too short, thereby limiting joining efficiency.
To address this limitation, a research team led by Professor Hiroshi Abe and Assistant Professor Masahito Inagaki at Nagoya University , in collaboration with Professor Natsuhisa Oka at Gifu University , studied DNA cleavage at targeted sites using chemical reactions instead of restriction enzymes.
The researchers examined a reaction reported between 1990 and 1992, in which silver ions cleave 3′-thiol-modified DNA at specific sites. They assessed its potential to generate suitable sticky ends. Results showed that although silver ions efficiently cleave DNA, they also bind nonspecifically, leading to precipitation. This resulted in a low DNA recovery rate of about 14%, which is insufficient for practical use.
The team then employed silver nanoparticles instead, hypothesizing that these could be removed after the reaction through centrifugation, thereby potentially increasing DNA recovery.
Experiments showed that DNA-cleaving efficiency reached about 50% at 70°C and nearly 100% at 95°C within two hours. However, these high temperatures pose a risk of damaging long-chain DNA.
To address this, the team coated the nanoparticles with polyethylene glycol (PEG), a water-soluble polymer, to enhance stability and dispersion. This modification increased cleaving efficiency from 36% without PEG to 92% with PEG at 37°C over 31 hours. “In the end, we optimized the conditions to a practical level and, under ambient temperatures, achieved PEG-modified cleaving efficiency above 91% at 50°C within just one to two hours,” stated Inagaki, the study’s first author.
An additional benefit of this process was the removal of unwanted DNA fragments bound to nanoparticle surfaces, leaving only the desired fragments with sticky ends in solution. This purification process increased the final DNA recovery rate from 14% to 98%.
The use of silver nanoparticles also enabled the generation of DNA fragments with 8-base sticky ends, a process that is challenging with conventional restriction enzymes. By employing T4 DNA ligase to join these fragments, the team achieved about double the joining efficiency of traditional methods. With an 18-base overhang, joining efficiency reached 44%, compared to only 8% with a conventional 4-base overhang, representing a fivefold improvement.
To evaluate the practical application of this approach, the researchers assembled a DNA fragment encoding green fluorescent protein (GFP) and introduced it into human HeLa cells. They successfully confirmed GFP expression, indicating accurate assembly.
Inagaki commented, “We believe this technology will be useful for synthesizing genomic DNA, with many possible applications in areas such as mRNA library establishment for cancer vaccines and gene therapy, as well as the development of artificial protein drugs and genome crops.”
He also explained the next step: “We have shown that two DNA fragments can be joined. Now, we need to confirm whether multiple fragments can be joined at the same time—a key step for building genome-scale DNA.”