Cyclodextrins as eminent constituents in nanoarchitectonics for drug delivery systems.

Cyclodextrins have been widely employed for drug delivery systems (DDSs) in which drugs are selectively delivered to a target site in the body. Recent interest has been focused on the construction of cyclodextrin-based nanoarchitectures that show sophisticated DDS functions. These nanoarchitectures are precisely fabricated based on three important features of cyclodextrins, namely (1) the preorganized three-dimensional molecular structure of nanometer size, (2) the easy chemical modification to introduce functional groups, and (3) the formation of dynamic inclusion complexes with various guests in water. With the use of photoirradiation, drugs are released from cyclodextrin-based nanoarchitectures at designated timing. Alternatively, therapeutic nucleic acids are stably protected in the nanoarchitectures and delivered to the target site. The efficient delivery of the CRISPR-Cas9 system for gene editing was also successful. Even more complicated nanoarchitectures can be designed for sophisticated DDSs. Cyclodextrin-based nanoarchitectures are highly promising for future applications in medicine, pharmaceutics, and other relevant fields.

Affinity Isolation of Defined Genomic Fragments Cleaved by Nuclease S1-based Artificial Restriction DNA Cutter.

The human genome is highly susceptible to various modifications, lesions, and damage. To analyze lesions and proteins bound to a defined region of the human genome, the genome should be fragmented at desired sites and the region of interest should be isolated. The few available methods for isolating a desired region of the human genome have serious drawbacks and can only be applied to specific sequences or require tedious experimental procedures. We have recently developed a novel method to isolate a desired fragment of the genome released by site-specific scission of DNA using a pair of pseudo-complementary peptide nucleic acids (pcPNAs) and S1 nuclease. When conjugated to biotin, one of the pcPNAs can be used to affinity purify the cleavage product. Here we report a detailed protocol to isolate defined kilobase-length DNA fragments that can be applied to plasmid or genomic DNA and is not limited by sequence. © 2019 by John Wiley & Sons, Inc.

Site-selective scission of human genome using PNA-based artificial restriction DNA cutter.

Site-selective scission of genomes is quite important for future biotechnology. However, naturally occurring restriction enzymes cut these huge DNAs at too many sites and cannot be used for this purpose. Recently, we have developed a completely chemistry-based artificial restriction DNA cutter (ARCUT) by combining a pair of pseudo-complementary PNA (pcPNA) strands (sequence recognition moiety) and Ce(IV)/EDTA complex (molecular scissors). The scission site of ARCUT and its scission specificity can be freely modulated in terms of the sequences and lengths of the pcPNA strands so that even huge genomes can be selectively cut at only one predetermined site. In this chapter, the method of site-selective scission of human genomic DNA using ARCUT is described in detail.

PNA-NLS conjugates as single-molecular activators of target sites in double-stranded DNA for site-selective scission.

Artificial DNA cutters have been developed by us in our previous studies by combining two strands of pseudo-complementary peptide nucleic acid (pcPNA) with Ce(IV)-EDTA-promoted hydrolysis. The pcPNAs have two modified nucleobases (2,6-diaminopurine and 2-thiouracil) instead of conventional A and T, and can invade double-stranded DNA to activate the target site for the scission. This system has been applied to site-selective scissions of plasmid, λ-phage, E. coli genomic DNA, and human genomic DNA. Here, we have reported a still simpler and more convenient DNA cutter obtained by conjugating peptide nucleic acid (PNA) with a nuclear localization signal (NLS) peptide. This new DNA cutter requires only one PNA strand (instead of two) bearing conventional (non-pseudo-complementary) nucleobases. This PNA-NLS conjugate effectively activated the target site in double-stranded DNA and induced site-selective scission by Ce(IV)-EDTA. The complex formation between the conjugate and DNA was concretely evidenced by spectroscopic results based on time-resolved fluorescence. The target scission site of this new system was straightforwardly determined by the Watson-Crick base pairing rule, and mismatched sequences were clearly discriminated. Importantly, even highly GC-rich regions, which are difficult to be targeted by a previous strategy using pcPNA, were successfully targeted. All these features of the present DNA cutter make it promising for various future applications.

Intracellular localization of PNA in human cells upon its introduction by electroporation.

Peptide nucleic acid (PNA) is one of the most useful DNA analogs in a wide variety of gene analysis in human cells. In order to exhibit its maximal functions, PNA must be localized to a desired place (e.g., nucleus, cytoplasm and other organelles). Here, we introduced PNAs into HeLa cells by electroporation and examined their localization at various time points. The PNA which binds to the mitochondrial COII gene was initially accumulated in the nucleus, and thereafter mostly transferred to cytoplasm. This time-dependent intracellular localization of PNA is ascribed to the breakdown of the nuclear envelope in the cell division. On the other hand, another PNA that binds to telomere repeat sequence mostly remained in the nucleus, even after the cell division occurred. The retention of this PNA in the nucleus was further enhanced when it was conjugated with Cy3.

Chemical and biological approaches to improve the efficiency of homologous recombination in human cells mediated by artificial restriction DNA cutter.

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.

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.

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.

Mismatch recognition in PNA double-duplex invasion.

Double-duplex invasion of pseudo-complementary PNA (pcPNA) to double-stranded DNA is promising for recognition of a specific sequence in double-stranded DNA. In order to apply this process for various purposes such as gene suppression, one base-pair change at the target site in DNA must be strictly distinguished by the pcPNA additives. In this study, mismatch-recognizing activity of double-duplex invasion was investigated under various salt conditions. It has been found that the mismatch-recognition of the invasion is strict enough to distinguish one base-pair alternation, as long as the invasion is achieved in the media of appropriate ionic strength (e.g., [NaCl] = 20 mM).

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.

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.

Highly efficient strand invasion by peptide nucleic acid bearing optically pure lysine residues in its backbone.

Chiral PNA monomers (PNA = peptide nucleic acid), in which nucleobases are attached to N-(aminoethyl)-D-lysine, were introduced to PNAs bearing pseudo-complementary nucleobases (2,6-diaminopurine and 2-thiouracil). When these highly cationic PNAs targeted double-stranded DNA, they invaded there much more efficiently than conventional pseudo-complementary PNAs composed of achiral PNA monomers. Although introduction of N-(aminoethyl)-D-lysine backbone was effective for promotion of strand invasion, L-isomer never promote it. Simple incorporation of lysine groups to the termini of PNA was also ineffective, indicating that introduction of positive charges into PNA backbone is important. Even highly G-C rich sequence, which conventional pseudo-complementary PNAs never invade, was successfully targeted based on this strategy.

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.

Rapid site-selective hydrolysis of double-stranded DNA by use of Ce(IV)/EDTA and PNA bearing phosphate group.

In order to hydrolyze double-stranded DNA efficiently at the target site, two pseudo-complementary peptide nucleic acids (pcPNAs) bearing phosphate group were combined with Ce(IV)/EDTA complex (EDTA = ethylenediamine-N,N,N',N'-tetraacetate). The phosphate groups as metal-binding ligands were placed near the target site, and concentrated the Ce(IV) complex thereto. As the result, the site-selective hydrolysis was notably promoted, compared with the scission by the cutters involving unmodified pcPNAs.

Construction of chimera protein by using artificial restriction DNA cutter.

We have already developed artificial restriction DNA cutter (ARCUT), which can hydrolyze double-stranded DNA site-selectively, by using Ce(IV)/EDTA in combination with two pseudo-complementary peptide nucleic acids (pcPNAs). Here, ARCUT was used to prepare a chimera protein. The gene for WW-domain containing oxidoreductase (WWOX) was clipped off by ARCUT just before its stop codon, and ligated with the gene for enhanced green fluorescent protein (EGFP). Conventional PCR for insertion of restriction enzyme site is never required.

Site-selective and hydrolytic two-strand scission of double-stranded DNA using Ce(IV)/EDTA and pseudo-complementary PNA.

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 degrees 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.

Combination of S1 nuclease and PNA for site-selective hydrolysis of double-stranded DNA. Comparison with the site-selective hydrolysis using Ce(IV)/EDTA.

The potential of the combination of SI nuclease and pseudo-complementary PNA (pcPNA) for site-selective scission of double-stranded DNA has been investigated. Through strand invasion of two pcPNAs, single-stranded portions were formed in both strands of substrate DNA. In the initial stage of the enzymatic digestion, two scission fragments were obtained due to the hydrolysis at these two gap-like sites. On prolonged reactions, however, these products (as well as the substrate DNA) were further digested to smaller fragments. Under the conditions employed here, only Ce(IV)/EDTA is available for the preparation of desired fragments from double-stranded DNA.

Manipulation of double-stranded DNA by artificial restriction enzyme composed of Ce(IV)/EDTA and PNA.

Through the invasion of pseudo-complementary PNA (pePNA) to double-stranded DNA, gap-like structures were formed at predetermined sites in both strands of PBR322 plasmid DNA. These gap-like sites were selectively hydrolyzed by Ce(IV)/EDTA complex, and two designed fragments were obtained. Furthermore, the scission fragment by this artificial restriction enzyme was successfully ligated with foreign DNA.