Artificial site-specific DNA-nicking system based on common restriction enzyme assisted by PNA openers

We report on the peptide nucleic acid (PNA)-directed design of a DNA-nicking system that enables selective and quantitative cleavage of one strand of duplex DNA at a designated site, thus mimicking natural nickases and significantly extending their potential. This system exploits the ability of pyrimidine PNAs to serve as openers for specific DNA sites by invading the DNA duplex and exposing one DNA strand for oligonucleotide hybridization. The resultant secondary duplex can act as a substrate for a restriction enzyme, which ultimately creates a nick in the parent DNA. We demonstrate that several restriction enzymes of different types could be successfully used in the PNA-assisted system we developed. Importantly, the enzyme cleavage efficiency is basically not impaired on such artificially generated substrates, compared with the efficiency on regular DNA duplexes. Our design originates a vast class of semisynthetic rare-cleaving DNA nickases, which are essentially absent at present. In addition, we show that the site-specific PNA-assisted nicking of duplex DNA can be engaged in a rolling-circle DNA amplification (RCA) reaction. This new RCA format demonstrates the practical potential of the novel biomolecular tool we propose for DNA technology and DNA diagnostics.