Abstract
DNA manipulations using a completely chemistry-based DNA cutter (ARCUT) have been reviewed. This cutter, recently developed by the authors, is composed of Ce(IV)/EDTA complex and two strands of pseudo-complementary peptide nucleic acid. The site-selective scission proceeds via hydrolysis of targeted phosphodiester linkages, so that the resultant scission fragments can be easily ligated with other fragments by using DNA ligase. Importantly, scission-site and site-specificity of the cutter are freely tuned in terms of the Watson–Crick rule. Thus, when one should like to manipulate DNA according to the need, he or she does not have to think about (1) whether appropriate “restriction enzyme sites” exist near the manipulation site and (2) whether the site-specificity of the restriction enzymes, if any, are sufficient to cut only the aimed position without chopping the DNA at non-targeted sites. Even the human genome can be manipulated, since ARCUT can cut the genome at only one predetermined site. Furthermore, the cutter is useful to promote homologous recombination in human cells, converting a site to desired sequence. The ARCUT-based DNA manipulation should be promising for versatile applications.
Highlights
Current biotechnology is primarily based on cut-and-paste of plasmid DNA in which restriction enzymes and DNA ligase are used
All the components required for homologous recombination (two pseudo-complementary peptide nucleic acid (pcPNA), Ce(IV)/EDTA and enhanced green fluorescent protein (EGFP) donor fragment) were incorporated by electroporation
We recently developed a completely chemistry-based DNA cutter (ARCUT) which selectively cuts double-stranded DNA at designated site with desired specificity
Summary
Current biotechnology is primarily based on cut-and-paste of plasmid DNA in which restriction enzymes and DNA ligase are used. Our group prepared an artificial restriction DNA cutter (ARCUT), which is completely chemistry-based and cuts double-stranded DNA at the desired site with aimed site-specificity [1,2,3,4,5,6,7]. There, DNA-cutting module of naturally occurring nucleases (or the catalytic center of restriction enzymes) is conjugated with other proteins, which selectively bind to the target sequence in DNA (e.g., zinc finger proteins and transcription activator-like effector proteins) The developments of these protein-based cutters are remarkable in this decade, and they are highly promising for future applications, especially for gene targeting in cells. The local concentration of the molecular scissors at the target site is increased and the site-selective DNA scission is remarkably promoted Throughout this review, this type of ARCUT (Ce(IV)/EDTA + two strands of pcPNA bearing a monophosphate) will be used
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