Artificial restriction DNA cutters (ARCUT) for future biotechnology

Recent chemical approaches towards artificial restriction DNA cutters (ARCUT) are reviewed. New tools to cut either single-stranded DNA or double-stranded DNA at predetermined sites have been prepared. As molecular scissors, these cutters employ Ce(IV)/EDTA complex that preferentially hydrolyzes single-stranded DNA over double-stranded DNA. Hot-spots for site-selective scission are prepared at predetermined site with the use of appropriate DNA or peptide nucleic acid (PNA) additives. Thus, gap-structures are formed in single-stranded DNA using two oligonucleotide additives, whereas invasion of a pair of PNAs is used for the scission of double-stranded DNA. These single-stranded portions are hydrolyzed by Ce(IV)IEDTA, resulting in the site-selective scission. The DNA scission proceeds totally via hydrolysis of phosphodiester linkages. Both linear DNA and supercoiled DNA are selectively cut at the target site by these restriction DNA cutters, and the fragments are efficiently combined with foreign DNA by using ligase. The recombinant DNA is successfully expressed in cells and produces the target protein (e.g., green fluorescent protein and other fusion proteins). No undesired side-reactions concurrently take place during the DNA manipulation. The length and the sequence of the recognition sites of these man-made DNA cutters are freely chosen, and thus, in principle, even huge DNA of higher animals and higher plants can be selectively cut and manipulated.