Cut DNA Research Articles

Artificial Restriction DNA Cutters to Promote Homologous Recombination in Human Cells

Homologous recombination is almost the only way to modify the genome in a predetermined fashion, despite its quite low frequency in mammalian cells. It has been already reported that the frequency of this biological process can be notably increased by inducing a double strand break (DSB) at target site. This article presents completely chemistry-based artificial restriction DNA cutter (ARCUT) for the promotion of homologous recombination in human cells. This cutter is composed of Ce(IV)/EDTA complex (molecular scissors) and two strands of peptide nucleic acid (PNA), and contains no proteins. Its scission site in the genome is determined simply by Watson-Crick rule so that ARCUT for desired homologous recombination is easily and straightforwardly designed and synthesized. The site-specificity of the scission is high enough to cut human genome at one target site. The DSB induced by this cutter is satisfactorily recognized by the repair system in human cells and promotes the targeted homologous recombination.

Artificial DNA cutters for DNA manipulation and genome engineering

This tutorial review provides recent developments in artificial cutters for site-selective scission of DNA with the focus on chemistry-based DNA cutters. They are useful tools for molecular biology and biotechnology, since their site-selectivity of scission is much higher than that of naturally occurring restriction enzymes and also their scission site is freely chosen. In order to prepare these cutters, a DNA-cutting molecule is combined with a sequence-recognizing molecule in a covalent or non-covalent way. At targeted sites in single-stranded and double-stranded DNAs, the scission occurs via either oxidative cleavage of nucleotides or hydrolysis of phosphodiester linkages. Among many successful examples, an artificial restriction DNA cutter, prepared from Ce(iv)/EDTA and pseudo-complementary peptide nucleic acid, hydrolyzed double-stranded DNA at the target site. The scission site and scission specificity are determined simply in terms of the Watson-Crick rule so that even the whole genome of human beings was selectively cut at one predetermined site. Consistently, homologous recombination in human cells was successfully promoted by this tool. For the purpose of comparison, protein-based DNA cutters (e.g., zinc finger nucleases) are also briefly described. The potential applications of these cutters and their future aspects are discussed.

Site-selective DNA hydrolysis induced by a metal-free peptide nucleic acid–cyclen conjugate

A metal-free artificial restriction DNA cutter which is composed of cyclen and classical peptide nucleic acid (PNA) was synthesized. Analysis of DNA cleavage products indicates the site-selective hydrolysis.

ChemInform Abstract: Artificial Restriction DNA Cutters as New Tools for Gene Manipulation

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Artificial Restriction DNA Cutters as New Tools for Gene Manipulation

The final cut. Two types of artificial tools (artificial restriction DNA cutter and zinc finger nuclease) that cut double-stranded DNA through hydrolysis of target phosphodiester linkages, have been recently developed. The chemical structures, preparation, properties, and typical applications of these two man-made tools are reviewed.Two types of artificial tools that cut double-stranded DNA through hydrolysis of target phosphodiester linkages have been recently developed. One is the chemistry-based artificial restriction DNA cutter (ARCUT) that is composed of a Ce(IV)-EDTA complex, which catalyses DNA hydrolysis, and a pair of pseudo-complementary peptide nucleic acid fragments for sequence recognition. Another type of DNA cutter, zinc finger nuclease (ZFN), is composed of the nuclease domain of naturally occurring FokI restriction endonuclease and a designed zinc finger DNA-binding domain. For both of these artificial tools, the scission site and specificity can be freely chosen according to our needs, so that even huge genomic DNA sequences can be selectively cut at the target site. In this article, the chemical structures, preparation, properties, and typical applications of these two man-made tools are described.

Origin of High Fidelity in Target-Sequence Recognition by PNA−Ce(IV)/EDTA Combinations as Site-Selective DNA Cutters

Double-duplex invasion of pseudocomplementary peptide nucleic acid (pcPNA) is one of the most important strategies for recognizing a specific site in double-stranded DNA (Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11804-11808). This strategy has recently been used to develop artificial restriction DNA cutters (ARCUTs) for site-selective scission of double-stranded DNA, in which a hot spot formed by double-duplex invasion of PNA was hydrolyzed by Ce(IV)/EDTA (Nat. Protoc. 2008, 3, 655-662). The present paper shows how and where the target sequence in double-stranded DNA is recognized by the PNA-Ce(IV)/EDTA combinations for site-selective scission. The mismatch-recognizing activities in both the invasion process and the whole scission process are evaluated. When both pcPNA additives are completely complementary to each strand of the DNA, site-selective scission is the most efficient, as expected. Upon exchange of one DNA base pair at the invasion site with another base pair, which introduces mismatches between the pcPNAs and the DNA, the site-selective scission by the ARCUT is notably diminished. Mismatches in (or near) the central double-invasion region are especially fatal, showing that Watson-Crick pairings of the DNA bases in this region with the pcPNA strands are essential for precise recognition of the target sequence. Both gel-shift assays and melting temperature measurements on the double-duplex invasion process have confirmed that the fidelity in this process primarily governs the fidelity of the DNA scission. According to these systematic analyses, the typical ARCUT involving two 15-mer pcPNAs precisely recognizes 14-16 base pairs in substrate DNA. This remarkable fidelity is accomplished at rather high salt concentrations that are similar to the values in cells.

Homologous recombination in human cells using artificial restriction DNA cutter

The double strand break induced by an artificial restriction DNA cutter (ARCUT) was successfully repaired in human cells with high frequencies through homologous recombination.

Site-selective scission of human genome by artificial restriction DNA cutter

By using an artificial restriction DNA cutter which is composed of Ce(iv)/EDTA and two pseudo-complementary peptide nucleic acid strands (pcPNAs), only one target site in the whole genome of human beings (one site in the X chromosome) was selectively hydrolyzed.

Site-specific gene manipulation of fluorescent proteins using artificial restriction DNA cutter

Two of three amino acid residues, which compose the chromophore of the enhanced green fluorescent protein (EGFP), were converted to others by using artificial restriction DNA cutter (ARCUT). The vector prepared by ARCUT was easily connected with the insert by using oligonucleotide additive and resultant fluorescent protein such as blue fluorescent protein (BFP) was successfully expressed in cells.

Site‐Selective Blocking of PCR by a Caged Nucleotide Leading to Direct Creation of Desired Sticky Ends in The Products

In order to terminate the polymerase reaction at a desired position, a caged thymine derivative--4-O-[2-(2-nitrophenyl)propyl]thymine--was incorporated into PCR primers. In the PCR cycles, the elongation of the nascent strand (5'-->3' direction) by polymerase was site-selectively terminated at the 3'-side of T(NPP). Accordingly, predetermined protruding ends were obtained after the removal of the protecting group by short UVA irradiation. Recombinant vectors coding the GFP gene were successfully prepared by direct ligation of these light-assisted cohesive-ending PCR (LACE-PCR) products with scission fragments obtained by use either of restriction enzymes or of artificial restriction DNA cutters and were used for transformation of E. coli.

Site-specific gene cassette insertion by combining artificial restriction DNA cutter and single-stranded DNA specific endonuclease

An artificial restriction DNA cutter (ARCUT), which was composed of Ce(IV)/EDTA complex and a pair of pseudo-complementary peptide nucleic acid, was used for construction of recombinant plasmid vector. The plasmid vector was cut by ARCUT only at target site, and resultant specific overhangs were blunted by treatment with Mung Bean Nuclease and Klenow fragment. This fragment having blunt ends at both termini was ligated with the gene of enhanced green fluorescent protein (EGFP) prepared by PCR. The resultant recombinant vector was translated and the fluorescent protein was successfully expressed in cells. Throughout the manipulation, neither natural restriction enzymes nor homologous recombination was employed. Thus, this strategy has promising applicability to construction of still larger vectors.

Artificial restriction DNA cutter for site-selective scission of double-stranded DNA with tunable scission site and specificity

The artificial restriction DNA cutter (ARCUT) method to cut double-stranded DNA at designated sites has been developed. The strategy at the base of this approach, which does not rely on restriction enzymes, is comprised of two stages: (i) two strands of pseudo-complementary peptide nucleic acid (pcPNA) anneal with DNA to form 'hot spots' for scission, and (ii) the Ce(IV)/EDTA complex acts as catalytic molecular scissors. The scission fragments, obtained by hydrolyzing target phosphodiester linkages, can be connected with foreign DNA using DNA ligase. The location of the scission site and the site-specificity are almost freely tunable, and there is no limitation to the size of DNA substrate. This protocol, which does not include the synthesis of pcPNA strands, takes approximately 10 d to complete. The synthesis and purification of the pcPNA, which are covered by a related protocol by the same authors, takes an additional 7 d, but pcPNA can also be ordered from custom synthesis companies if necessary.

Development of Artificial Restriction DNA Cutter Composed of Ce(Iv)/EDTA and PNA

An artificial restriction enzyme, which we developed recently by combining Ce(IV)/EDTA and peptide nucleic acids, was used for PCR-free construction of a fusion protein. The fusion protein was successfully expressed in mammalian cells. This artificial DNA cutter can be also applied to site-selective scission of huge DNAs. Promising features of this novel tool were concretely evidenced.

Gene manipulation of adenovirus vector by artificial restriction DNA cutter

Adenovirus vector is a useful tool for research in gene expression and regulation as well as gene therapy. However, the size of this vector is too large to construct it by a simple method. In this study, we successfully cleaved specifically at one target site of this large vector by artificial restriction DNA cutter (ARCUT). This result suggests that ARCUT will be the powerful tool for construction of large sized virus vectors.

Ce(IV)-Catalyzed Site-Selective DNA Hydrolysis Using Tris- and Tetrakismethylenephosphonate-Modified Oligonucleotides

To improve the recruitment of the catalytically active Ce(IV) ion to the DNA cleavage site in the ARCUT (Artificial Restriction DNA Cutter) system, two phosphonate ligands, nitrilotris(methylenephosphonic acid) (NTP) and N,N,N',N'-ethylenediaminetetra-(methylenephosphonic acid) (EDTP), have been linked to the 5'- or 3'-end of oligonucleotides complementary to the target DNA. Compared to their counterparts bearing a terminal monophosphate, these new modified oligonucleotides exhibit a modest to significant increase in the cleavage activity, depending on the target.

Activation of Double-stranded DNA by One pcPNA Strand for Its Site-selective Scission with CeIV/EDTA

Abstract In the presence of only one strand of pseudo-complementary PNA (pcPNA), double-stranded DNA was selectively hydrolyzed by CeIV/EDTA (EDTA = ethylenediamine-N,N,N′,N′-tetraacetate) at target site. Compared with previously reported artificial restriction DNA cutters using two pcPNA strands, the present cutters are simple and promising for various applications.

Chemical modification of Ce(IV)/EDTA-based artificial restriction DNA cutter for versatile manipulation of double-stranded DNA

A monophosphate group was attached to the terminus of pseudo-complementary peptide nucleic acid (pcPNA), and two of thus modified pcPNAs were combined with Ce(IV)/EDTA for site-selective hydrolysis of double-stranded DNA. The site-selective DNA scission was notably accelerated by this chemical modification of pcPNAs. These second-generation artificial restriction DNA cutters (ARCUTs) differentiated the target sequence so strictly that no scission occurred even when only one DNA base-pair was altered to another. By using two of the activated ARCUTs simultaneously, DNA substrate was selectively cut at two predetermined sites, and the desired fragment was clipped and cloned. The DNA scission by ARCUT was also successful even when the target site was methylated by methyltransferase and protected from the corresponding restriction enzyme. Furthermore, potentiality of ARCUT for manipulation of huge DNA has been substantiated by site-selective scission of genomic DNA of Escherichia coli (composed of 4,600,000 bp) at the target site. All these results indicate promising applications of ARCUTs for versatile purposes.

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)/EDTA, 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. Keywords: DNA hydrolysis, Oligonucleotide Additives, Gene Manipulation, pcPNA additives, Blue Fluorescence, GFP gene

Highly active artificial restriction enzyme composed of Ce(IV)/EDTA and PNA bearing phosphate group - Relationship between the promotion by phosphate and the structure of invasion complex -

Recently, we developed artificial restriction DNA cutter (ARCUT) composed of pseudo-complementary peptide nucleic acid (pcPNA) and Ce(IV)/EDTA complex (EDTA = ethylenediamine-N,N,N',N'-tetraacetate). Here we promoted the site-selective hydrolysis by attaching phosphate groups to the pcPNAs. The promotion by the phosphates increased with decreasing length of the gap-like site. Furthermore, the scission was successful even when phosphate groups were introduced to 0 base-gap system.

Site-selective hydrolysis of huge DNA by artificial restriction DNA cutter

Genomic DNAs of lambda phage (48.5 kbp) and E. coli (4.6 Mbp) were successfully cleaved at the target site by artificial restriction DNA cutter (ARCUT), which we recently developed by combining Ce(IV)/EDTA and PNA (peptide nucleic acid). PNA satisfactorily invaded the target site in such huge DNAs and activate there for Ce(IV)/EDTA-induced scission.