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 2 to 5x higher DNA assembly efficiency than conventional restriction enzyme methods.
In the study, published in Nucleic Acids Research, researchers adapted a reaction reported in the late-90s that used silver ions cleave DNA at specific sites. In this modern version, the researchers employed silver nanoparticles instead, hypothesizing that these could be removed after the reaction through centrifugation, 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.
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%.
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.
Now that the researchers have shown that two DNA fragments can be joined, the next step is to confirm whether multiple fragments can be joined at the same time—a key step for building genome-scale DNA.
Data from Nagoya University