A Japanese research group announced on June 10 the development of a silver nanoparticle-based technology that enables precise cutting and joining of DNA at targeted sites, resulting in two to five times higher DNA assembly efficiency compared to conventional restriction enzyme methods. The findings were published in the journal Nucleic Acids Research.
DNA is composed of long chains that serve as blueprints for living organisms. In genetic engineering, scientists cut DNA at specific sites and join the resulting fragments to other sequences, which allows applications such as advanced crop breeding, genetic disease treatment, and animal model generation for drug discovery.
Traditional methods use restriction enzymes to cut DNA and T4 DNA ligase to reconnect fragments. However, these enzymes only work at certain sequences and often produce sticky ends that are too short for efficient joining. To address this limitation, a team led by Professor Hiroshi Abe and Assistant Professor Masahito Inagaki at Nagoya University, with Professor Natsuhisa Oka at Gifu University, studied chemical reactions using silver ions instead of restriction enzymes. While silver ions could cleave 3'-thiol-modified DNA efficiently, they also caused nonspecific binding and precipitation that resulted in low recovery rates.
The researchers then used silver nanoparticles instead of ions, hypothesizing they could be removed after reaction through centrifugation to improve recovery. Experiments showed high cleavage efficiency but required elevated temperatures that risked damaging long-chain DNA. By coating nanoparticles with polyethylene glycol (PEG), they increased stability and dispersion; this raised 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," said Inagaki.
An added benefit was purification: unwanted fragments bound to nanoparticle surfaces were removed during processing while desired sticky-ended fragments remained in solution—raising final recovery from 14% up to 98%. The approach enabled generation of longer sticky ends (up to eight bases), which is difficult with conventional enzymes; joining these with T4 ligase achieved about double or even fivefold higher efficiency than traditional four-base overhangs depending on fragment length.
To test practical application, the team assembled a green fluorescent protein (GFP) gene fragment using their method and introduced it into human HeLa cells—successfully confirming GFP expression. "We believe this technology will be useful for synthesizing genomic DNA...in areas such as mRNA library establishment for cancer vaccines and gene therapy," said Inagaki. He added, "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."
