DNA consists of long molecular chains that contain the genetic instructions essential for life. In genetic engineering, scientists cut DNA at precise locations and combine the resulting fragments with other genetic sequences. This DNA assembly process supports many applications, including crop improvement, gene therapy, genetic disease research, and the development of animal models for drug discovery.
To join short DNA fragments efficiently, researchers use exposed sequences known as sticky ends. These complementary overhangs allow DNA fragments to bind together before they are permanently joined. However, producing sticky ends at precisely selected locations remains challenging because many existing DNA-cutting technologies have limited sequence flexibility.
Japanese researchers have developed a new DNA assembly method that uses silver nanoparticles to cut and reconnect DNA at targeted sites. The technique produced DNA construction efficiencies two to five times higher than those achieved with conventional restriction enzyme methods. The study was published in Nucleic Acids Research.
Limitations of traditional DNA assembly methods
Conventional methods for assembling long DNA molecules generally rely on restriction enzymes to cut DNA and T4 DNA ligase to join the resulting fragments. However, restriction enzymes recognize and cut only specific DNA sequences. They also typically generate short sticky ends, which can limit the efficiency and stability of DNA fragment assembly.
Seeking a more flexible alternative, a research team led by Professor Hiroshi Abe and Assistant Professor Masato Inagaki of Nagoya University, together with Professor Natsuhisa Oka of Gifu University, investigated whether chemical reactions could cut DNA at selected positions without using restriction enzymes.
The researchers revisited a reaction first reported between 1990 and 1992, in which silver ions cut DNA modified with 3′-thiol groups at specific sites. They examined whether this reaction could be used to create functional sticky ends. Although silver ions successfully cleaved the DNA, they also bound nonspecifically and caused precipitation. Consequently, only about 14% of the DNA could be recovered, making the approach impractical for efficient DNA construction.
Silver nanoparticles increase DNA recovery
The team replaced silver ions with silver nanoparticles. Because the nanoparticles could be separated from the reaction mixture by centrifugation, the researchers believed this approach would make it easier to recover the desired DNA fragments.
Initial tests showed that DNA cleavage reached approximately 50% at 70°C and nearly 100% at 95°C within two hours. However, these high temperatures can damage long DNA molecules, creating another challenge for practical DNA assembly.
To address this problem, the researchers coated the silver nanoparticles with polyethylene glycol (PEG), a water-soluble polymer that improves nanoparticle stability and dispersibility. The PEG coating increased DNA cleavage efficiency from 36% without PEG to 92% with PEG during a 31-hour reaction at 37°C. “Ultimately, we optimized the conditions to a practical level and achieved a PEG-modified cleavage efficiency of more than 91% within just one to two hours at 50°C,” said Inagaki, the study’s first author.
The silver nanoparticle method also provided an important purification benefit. Unwanted DNA fragments remained attached to the nanoparticle surfaces, while the desired fragments containing sticky ends stayed in the solution. This built-in separation process increased final DNA recovery from 14% to 98%.
Long sticky ends improve DNA fragment binding
Silver nanoparticles enabled the researchers to generate DNA fragments with eight-base sticky ends, which are difficult to produce with conventional restriction enzymes. When the fragments were joined using T4 DNA ligase, the DNA assembly efficiency was approximately twice as high as that of traditional methods.
The advantage increased with longer overhangs. The researchers achieved a conjugation efficiency of 44% using an 18-base sticky end. In comparison, the efficiency for a conventional four-base overhang was only 8%, giving the new method approximately five times greater efficiency.
To test the method in a biological setting, the team assembled DNA fragments encoding green fluorescent protein (GFP). They then introduced the assembled DNA into human HeLa cells. The cells successfully produced GFP, confirming that the DNA fragments had been assembled correctly and remained functional.
Potential applications in gene therapy and synthetic biology
Professor Inagaki said, “This technology is useful for synthesizing genomic DNA, and we believe it has potential applications in a variety of fields, including the construction of mRNA libraries for cancer vaccines and gene therapy, as well as the development of artificial protein drugs and genomic crops.”
The researchers are now investigating whether the technique can assemble more than two DNA fragments simultaneously. “We showed that we can join two DNA fragments. Next, we need to determine whether we can join multiple fragments at the same time. This is an important step toward building genome-scale DNA,” Inagaki explained.
This research was supported by the Japan Science and Technology Agency (JST) (JPMJCR18S1, JPMJCR23N1, JP25H00427, JP24H00737, JP22H02219, JP22K21346 International Leading Research), the Japan Agency for Medical Research and Development (AMED) (JP22gm0010008 (LEAP), JP25ak0101289, JP223fa827 (SCADA), JP243fa827032 (SCADA), JP23bm1223009, JP24ek0109697, JP25ama221315, JP25km0405209, JP25ama221230, and JP23fk0210133), and the Tanaka Kikinzoku Memorial Foundation (Precious Metals Research Grants 2021 Silver Award to M.I.). Funding for the open-access publication fee was provided by the Japan Science and Technology Agency.
Source: www.sciencedaily.com