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1.  Cut-and-Paste of DNA Using an Artificial Restriction DNA Cutter 
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.
doi:10.3390/ijms14023343
PMCID: PMC3588047  PMID: 23385238
site-selective scission; DNA cutter; Ce(IV)/EDTA; PNA; human genome; homologous recombination; restriction enzyme-free manipulation
2.  Introduction of multiphosphonate ligand to peptide nucleic acid for metal ion conjugation 
Artificial DNA, PNA & XNA  2012;3(2):73-79.
Peptide nucleic acid (PNA) is one of the most widely used synthetic DNA analogs. Conjugation of functional molecules to PNA is very effective to further widen its potential applications. For this purpose, here we report the synthesis of several ligand monomers and introduced them to PNA. These ligand-modified PNAs attract cerium ion and are useful for site-selective DNA hydrolysis. It should be noted that these ligands on PNA are also effective even under the conditions of invasion complex.
doi:10.4161/adna.20727
PMCID: PMC3429533  PMID: 22772037
cerium; DNA; hydrolysis; ligand; metal ion; peptide nucleic acid
3.  Chemical and biological approaches to improve the efficiency of homologous recombination in human cells mediated by artificial restriction DNA cutter 
Nucleic Acids Research  2012;40(11):e81.
A chemistry-based artificial restriction DNA cutter (ARCUT) was recently prepared from Ce(IV)/EDTA complex and a pair of pseudo-complementary peptide nucleic acids. This cutter has freely tunable scission-site and site specificity. In this article, homologous recombination (HR) in human cells was promoted by cutting a substrate DNA with ARCUT, and the efficiency of this bioprocess was optimized by various chemical and biological approaches. Of two kinds of terminal structure formed by ARCUT, 3′-overhang termini provided by 1.7-fold higher efficiency than 5′-overhang termini. A longer homology length (e.g. 698 bp) was about 2-fold more favorable than shorter one (e.g. 100 bp). When the cell cycle was synchronized to G2/M phase with nocodazole, the HR was promoted by about 2-fold. Repression of the NHEJ-relevant proteins Ku70 and Ku80 by siRNA increased the efficiency by 2- to 3-fold. It was indicated that appropriate combination of all these chemical and biological approaches should be very effective to promote ARCUT-mediated HR in human cells.
doi:10.1093/nar/gks185
PMCID: PMC3367209  PMID: 22362741
4.  Nanomechanical DNA origami 'single-molecule beacons' directly imaged by atomic force microscopy 
Nature Communications  2011;2:449-.
DNA origami involves the folding of long single-stranded DNA into designed structures with the aid of short staple strands; such structures may enable the development of useful nanomechanical DNA devices. Here we develop versatile sensing systems for a variety of chemical and biological targets at molecular resolution. We have designed functional nanomechanical DNA origami devices that can be used as 'single-molecule beacons', and function as pinching devices. Using 'DNA origami pliers' and 'DNA origami forceps', which consist of two levers ~170 nm long connected at a fulcrum, various single-molecule inorganic and organic targets ranging from metal ions to proteins can be visually detected using atomic force microscopy by a shape transition of the origami devices. Any detection mechanism suitable for the target of interest, pinching, zipping or unzipping, can be chosen and used orthogonally with differently shaped origami devices in the same mixture using a single platform.
DNA origami involves the folding of long single-stranded DNA into designed structures that may aid the development of useful nanomechanical DNA devices. In this study, DNA origami pliers and forceps are shown to undergo conformational changes on single-molecule binding.
doi:10.1038/ncomms1452
PMCID: PMC3265375  PMID: 21863016
5.  Sensitive RNA detection by combining three-way junction formation and primer generation-rolling circle amplification 
Nucleic Acids Research  2011;40(3):e22.
Recently, we developed a simple isothermal nucleic acid amplification reaction, primer generation-rolling circle amplification (PG-RCA), to detect specific DNA sequences with great sensitivity and large dynamic range. In this paper, we combined PG-RCA with a three-way junction (3WJ) formation, and detected specific RNA molecules with high sensitivity and specificity in a one-step and isothermal reaction format. In the presence of target RNA, 3WJ probes (primer and template) are designed to form a 3WJ structure, from which multiple signal primers for the following PG-RCA can be generated by repeating primer extension, nicking and signal primer dissociation. Although this signal primer generation is a linear amplification process, the PG-RCA exponentially can amplify these signal primers and thus even a very small amount of RNA specimen can be detected. After optimizing the structures of 3WJ probes, the detection limit of this assay was 15.9 zmol (9.55 × 103 molecules) of synthetic RNA or 143 zmol (8.6 × 104 molecules) of in vitro transcribed human CD4 mRNA. Further, the applicability of this assay to detect CD4 mRNA in a human mRNA sample was demonstrated.
doi:10.1093/nar/gkr909
PMCID: PMC3273829  PMID: 22127872
6.  Photoswitching of Site-Selective RNA Scission by Sequential Incorporation of Azobenzene and Acridine Residues in a DNA Oligomer 
Journal of Nucleic Acids  2011;2011:162452.
Photoresponsive systems for site-selective RNA scission have been prepared by combining Lu(III) ions with acridine/azobenzene dual-modified DNA. The modified DNA forms a heteroduplex with substrate RNA, and the target phosphodiester linkages in front of the acridine residue is selectively activated so that Lu(III) ion rapidly cleaves the linkage. Azobenzene residue introduced adjacent to the acridine residue acts as a photoresponsive switch, which triggers the site-selective scission upon UV irradiation. A trans isomer of azobenzene efficiently suppresses the scission, whereas the cis isomer formed by UV irradiation hardly affects the scission. As a result, 1.7–2.4-fold acceleration of the cleavage was achieved simply by irradiating UV for 3 min to the mixture prior to the reaction. Considering the yield of photoisomerization, the intrinsic activity of a cis isomer is up to 14.5-fold higher than that of the trans isomer.
doi:10.4061/2011/162452
PMCID: PMC3177363  PMID: 21941627
7.  Sensitive isothermal detection of nucleic-acid sequence by primer generation–rolling circle amplification 
Nucleic Acids Research  2008;37(3):e19.
A simple isothermal nucleic-acid amplification reaction, primer generation–rolling circle amplification (PG–RCA), was developed to detect specific nucleic-acid sequences of sample DNA. This amplification method is achievable at a constant temperature (e.g. 60°C) simply by mixing circular single-stranded DNA probe, DNA polymerase and nicking enzyme. Unlike conventional nucleic-acid amplification reactions such as polymerase chain reaction (PCR), this reaction does not require exogenous primers, which often cause primer dimerization or non-specific amplification. Instead, ‘primers’ are generated and accumulated during the reaction. The circular probe carries only two sequences: (i) a hybridization sequence to the sample DNA and (ii) a recognition sequence of the nicking enzyme. In PG–RCA, the circular probe first hybridizes with the sample DNA, and then a cascade reaction of linear rolling circle amplification and nicking reactions takes place. In contrast with conventional linear rolling circle amplification, the signal amplification is in an exponential mode since many copies of ‘primers’ are successively produced by multiple nicking reactions. Under the optimized condition, we obtained a remarkable sensitivity of 84.5 ymol (50.7 molecules) of synthetic sample DNA and 0.163 pg (∼60 molecules) of genomic DNA from Listeria monocytogenes, indicating strong applicability of PG–RCA to various molecular diagnostic assays.
doi:10.1093/nar/gkn1014
PMCID: PMC2647323  PMID: 19106144
8.  Chiral introduction of positive charges to PNA for double-duplex invasion to versatile sequences 
Nucleic Acids Research  2008;36(5):1464-1471.
Invasion of two PNA strands to double-stranded DNA is one of the most promising methods to recognize a predetermined site in double-stranded DNA (PNA = peptide nucleic acid). In order to facilitate this ‘double-duplex invasion’, a new type of PNA was prepared by using chiral PNA monomers in which a nucleobase was bound to the α-nitrogen of N-(2-aminoethyl)-d-lysine. These positively charged monomer units, introduced to defined positions in Nielsen's PNAs (poly[N-(2-aminoethyl)glycine] derivatives), promoted the invasion without impairing mismatch-recognizing activity. When pseudo-complementary nucleobases 2,6-diaminopurine and 2-thiouracil were bound to N-(2-aminoethyl)-d-lysine, the invasion successfully occurred even at highly G–C-rich regions [e.g. (G/C)7(A/T)3 and (G/C)8(A/T)2] which were otherwise hardly targeted. Thus, the scope of sequences available as the target site has been greatly expanded. In contrast with the promotion by the chiral PNA monomers derived from N-(2-aminoethyl)-d-lysine, their l-isomers hardly invaded, showing crucial importance of the d-chirality. The promotion of double-duplex invasion by the chiral (d) PNA monomer units was ascribed to both destabilization of PNA/PNA duplex and stabilization of PNA/DNA duplexes.
doi:10.1093/nar/gkm1154
PMCID: PMC2275137  PMID: 18203747
9.  Chemical modification of Ce(IV)/EDTA-based artificial restriction DNA cutter for versatile manipulation of double-stranded DNA 
Nucleic Acids Research  2007;35(7):e53.
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.
doi:10.1093/nar/gkm052
PMCID: PMC1874645  PMID: 17376805
10.  Site-selective and hydrolytic two-strand scission of double-stranded DNA using Ce(IV)/EDTA and pseudo-complementary PNA 
Nucleic Acids Research  2004;32(19):e153.
By combining Ce(IV)/EDTA with two pseudo-complementary peptide nucleic acids (pcPNAs), both strands in double-stranded DNA were site-selectively hydrolyzed at the target site. Either plasmid DNA (4361 bp) or its linearized form was used as the substrate. When two pcPNAs invaded into the double-stranded DNA, only the designated portion in each of the two strands was free from Watson–Crick base pairing with the counterpart DNA or the pcPNA. Upon the treatment of this invasion complex with Ce(IV)/EDTA at 37°C and pH 7.0, both of these single-stranded portions were selectively hydrolyzed at the designated site, resulting in the site-selective two-strand scission of the double-stranded DNA. Furthermore, the hydrolytic scission products were successfully connected with foreign double-stranded DNA by using ligase. The potential of these artificial systems for manipulation of huge DNA has been indicated.
doi:10.1093/nar/gnh151
PMCID: PMC528827  PMID: 15520462
11.  Straightforward detection of SNPs in double-stranded DNA by using exonuclease III/nuclease S1/PNA system 
Nucleic Acids Research  2004;32(4):e42.
Single-nucleotide polymorphisms (SNPs) in double-stranded DNA (dsDNA) have been straightforwardly genotyped by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS). Peptide nucleic acid (PNA), a DNA analog, was used as a probe molecule. In its presence, genomic dsDNA was first treated with exonuclease III and then with nuclease S1. By these one-pot reactions, single-stranded DNA fragments including the SNP sites were formed in situ. These fragments were directly analyzed by MALDI-TOF MS, and the identity of the DNA base at the SNP site was determined in terms of mass number. By using two or more PNA probes simultaneously, multiplex analysis was also successful. Various genotypes of apolipoprotein E gene (ε2/ε2, ε3/ε3, ε4/ε4, ε2/ε3 and ε3/ε4) were identified from dsDNA obtained by PCR from corresponding patients.
doi:10.1093/nar/gnh039
PMCID: PMC390314  PMID: 14982961
12.  Oligoamine–acridine conjugates for promotion of gap-selective DNA hydrolysis by Ce(IV)/EDTA complex 
Nucleic Acids Research  2003;31(15):4497-4502.
Oligoamines (spermidine, dipropylenetriamine and propylenediamine) were covalently attached to acridine via a hexamethylene linker. These oligoamine–acridine conjugates were efficiently bound to gap sites in substrate DNA, and promoted the DNA hydrolysis by a homogeneous Ce(IV)/ethylenediamine-N,N,N′,N′-tetraacetate (EDTA) complex at these sites. In contrast, the hydrolysis of the double-stranded portion in the DNA was little affected by these conjugates, although they were strongly bound thereto by the intercalation of their acridine moieties. As a result, the gap site was selectively and efficiently hydrolyzed by combining the Ce(IV)/EDTA complex with the oligoamine– acridine conjugate. Either the oligoamine or the acridine was only poorly active for the purpose, substantiating the essential role of cooperation between them. The promotion of gap-selective DNA hydrolysis by the conjugates has been ascribed to electrostatic stabilization of a negatively charged transition state by their positive charges.
PMCID: PMC169895  PMID: 12888510
13.  Preferential hydrolysis of gap and bulge sites in DNA by Ce(IV)/EDTA complex 
Nucleic Acids Research  2002;30(19):e102.
A new strategy for site-selective DNA hydrolysis, which takes advantage of the difference in reactivity between the phosphodiester linkages at the target site and the others, is presented. As the molecular scissors, homogeneous Ce(IV)/ethylenediamine-N,N,N′,N′-tetraacetate (EDTA) complex is used without being bound to any sequence-recognizing moiety. When a gap structure is formed at the target site by using two short oligonucleotides and the composite is treated with the Ce(IV)/EDTA complex at pH 7.0 and 37°C, the gap site in the substrate DNA is preferentially hydrolyzed over the double-stranded portion of the DNA. Site-selective DNA scission is also achieved by forming a bulge structure at the target site with the use of the appropriate oligonucleotide. These site-selective scissions are based on the following two factors: (i) the phosphodiester linkages in a single-stranded DNA are far more susceptible to the hydrolysis by the Ce(IV) complex than are the linkages in double-stranded DNA, and (ii) the phosphodiester linkages in the bulge sites are still more reactive than those in single-stranded DNA. In both cases, the addition of spermine significantly accelerates the scission.
PMCID: PMC140562  PMID: 12364619

Results 1-13 (13)