Silver nanoparticle-induced site-specific strand cleavage of chemically modified oligonucleotides for long-chain DNA assembly

Here, we report a novel site-specific oligonucleotide strand cleavage reaction mediated by silver nanoparticles (AgNPs) that is applicable for synthesizing sticky ends of PCR (polymerase chain reaction)-amplified DNA for DNA ligase-mediated ligation to build up long-chain DNA. For this purpose, we designed and synthesized modified DNA as a PCR primer bearing 3'-phosphorothiolate linkage at strand cleavage sites. The AgNP surface acts as a Lewis acid to activate the 3'-phosphorothiolate linkage of DNA and induces hydrolysis of the 3'-phosphorothiolate linkage to produce 3'-thiol-DNA and 5'-phosphorylated DNA fragments. In this study, we discuss the factors that induce oligonucleotide strand cleavage by AgNPs, such as nanoparticle size, reaction time, reaction temperature, and chemical modification of nanoparticles. Polyethylene glycol modification of AgNPs was found to maximize oligonucleotide strand cleavage activity. We demonstrated the synthesis of 848-bp DNA by DNA ligase-mediated ligation of 298- and 558-bp DNA fragments with sticky ends and eight bases overhang prepared using the AgNP-induced strand cleavage method. In addition, we successfully constructed a GFP-coding DNA and demonstrated the expression of GFP in HeLa cells. This research opens the possibility of developing new applications of metal nanoparticle chemistry for biotechnological tools.