Ce-based solid-phase catalysts for phosphate hydrolysis as new tools for next-generation nanoarchitectonics.

This review comprehensively covers synthetic catalysts for the hydrolysis of biorelevant phosphates and pyrophosphates, which bridge between nanoarchitectonics and biology to construct their interdisciplinary hybrids. In the early 1980s, remarkable catalytic activity of Ce4+ ion for phosphate hydrolysis was found. More recently, this finding has been extended to Ce-based solid catalysts (CeO2 and Ce-based metal-organic frameworks (MOFs)), which are directly compatible with nanoarchitectonics. Monoesters and triesters of phosphates, as well as pyrophosphates, were effectively cleaved by these catalysts. With the use of either CeO2 nanoparticles or elegantly designed Ce-based MOF, highly stable phosphodiester linkages were also hydrolyzed. On the surfaces of all these solid catalysts, Ce4+ and Ce3+ coexist and cooperate for the catalysis. The Ce4+ activates phosphate substrates as a strong acid, whereas the Ce3+ provides metal-bound hydroxide as an eminent nucleophile. Applications of these Ce-based catalysts to practical purposes are also discussed.

G-Quadruplexes in Human Telomere: Structures, Properties, and Applications.

G-quadruplexes, intricate four-stranded structures composed of G-tetrads formed by four guanine bases, are prevalent in both DNA and RNA. Notably, these structures play pivotal roles in human telomeres, contributing to essential cellular functions. Additionally, the existence of DNA:RNA hybrid G-quadruplexes adds a layer of complexity to their structural diversity. This review provides a comprehensive overview of recent advancements in unraveling the intricacies of DNA and RNA G-quadruplexes within human telomeres. Detailed insights into their structural features are presented, encompassing the latest developments in chemical approaches designed to probe these G-quadruplex structures. Furthermore, this review explores the applications of G-quadruplex structures in targeting human telomeres. Finally, the manuscript outlines the imminent challenges in this evolving field, setting the stage for future investigations.

Preferential production of RNA rings by T4 RNA ligase 2 without any splint through rational design of precursor strand.

Rings of single-stranded RNA are promising for many practical applications, but the methods to prepare them in preparative scale have never been established. Previously, RNA circularization was achieved by T4 RNA ligase 2 (Rnl2, a dsRNA ligase) using splints, but the yield was low due to concurrent intermolecular polymerization. Here, various functional RNAs (siRNA, miRNA, ribozyme, etc.) are dominantly converted by Rnl2 to the rings without significant limitations in sizes and sequences. The key is to design a precursor RNA, which is highly activated for the efficient circularization without any splint. First, secondary structure of target RNA ring is simulated by Mfold, and then hypothetically cut at one site so that a few intramolecular base pairs are formed at the terminal. Simply by treating this RNA with Rnl2, the target ring was selectively and efficiently produced. Unexpectedly, circular RNA can be obtained in high yield (>90%), even when only 2 bp form in the 3'-OH side and no full match base pair forms in the 5'-phosphate side. Formation of polymeric by-products was further suppressed by diluting conventional Rnl2 buffer to abnormally low concentrations. Even at high-RNA concentrations (e.g. 50 µM), enormously high selectivity (>95%) was accomplished.

Stepwise Strategy for One-Pot Synthesis of Single-Stranded DNA Rings from Multiple Short Fragments.

Cyclic rings of single-stranded (ss) DNA have various unique properties, but wider applications have been hampered by their poor availability. This paper reports a convenient one-pot method in which these rings are efficiently synthesized by using T4 DNA ligase through convergent cyclization of easily available short DNA fragments. The key to the present method is to separate all the splint oligonucleotides into several sets, and add each set sequentially at an appropriate interval to the solutions containing all the short DNA fragments. Compared with simple one-pot strategies involving simultaneous addition of all the splints at the beginning of the reaction, both the selectivity and the yields of target ssDNA rings are greatly improved. This convergent method is especially useful for preparing large-sized rings that are otherwise hard to obtain. By starting from six short DNA fragments (71-82 nt), prepared by a DNA synthesizer, a ssDNA ring of 452-nt size was synthesized in 35 mol % yield and in high selectivity. Satisfactorily pure DNA rings were obtainable simply by treating the crude products with exonuclease.

Facile Characterization of Topology of DNA Catenanes.

During the preparation of single-stranded DNA catenanes, topological isomers of different linking numbers (Lk) are intrinsically produced, and they must be separated from each other to construct sophisticated nanostructures accurately. In many previous studies, however, mixtures of these isomers were directly employed to construct nanostructures without sufficient characterization. Here, we present a method that easily and clearly characterizes the isomers by polyacrylamide gel electrophoresis. To the mixtures of topological isomers of [2]catenanes, two-strut oligonucleotides, which are complementary with a part of both rings, were added to connect the rings and fix the whole conformations of isomers. As a result, the order of migration rate was always Lk3 > Lk2 > Lk1, irrespective of gel concentration. Thus, all the topological isomers were unanimously characterized by only one polyacrylamide gel electrophoresis experiment. Well-characterized DNA catenanes are obtainable by this two-strut strategy, opening the way to more advanced nanotechnology.

Thermus thermophilus DNA Ligase Connects Two Fragments Having Exceptionally Short Complementary Termini at High Temperatures.

Thermus thermophilus DNA ligase (Tth DNA ligase) is widely employed for cloning, enzymatic synthesis, and molecular diagnostics at high temperatures (e.g., 65 °C). It has been long believed that the complementary ends must be very long (e.g., >30 bp) to place two DNA fragments nearby for the ligation. In the current study, the length of the complementary portion was systematically varied, and the ligation efficiency was evaluated using the high resolution melting (HRM) method. Unexpectedly, very short oligonucleotides (7-10 nt) were successfully ligated on the complementary overhang attached to a dsDNA at 70 °C. Furthermore, sticky ends with the overhang of only 4 nt long, available after scission with many restriction enzymes, were also efficiently ligated at 45-70 °C. The ligation yield for the 6-nt-long sticky ends was as high as 80%. It was concluded that Tth DNA ligase can be used as a unique tool for DNA manipulation that cannot be otherwise easily accomplished.

Effective Characterization of DNA Ligation Kinetics by High-Resolution Melting Analysis.

High-resolution melting (HRM) analysis has been improved and applied for the first time to quantitative analysis of enzymatic reactions. By using the relative ratios of peak intensities of substrates and products, the quantitativity of conventional HRM analysis has been improved to allow detailed kinetic analysis. As an example, the ligation of sticky ends through the action of T4 DNA ligase has been kinetically analyzed, with comprehensive data on substrate specificity and other properties having been obtained. For the first time, the kinetic parameters (kobs and apparent Km ) of sticky-end ligation were obtained for both fully matched and mismatched sticky ends. The effect of ATP concentration on sticky-end ligation was also investigated. The improved HRM method should also be applicable to versatile DNA-transforming enzymes, because the only requirement is that the products have Tm values different enough from the substrates.

Terminal hairpin in oligonucleotide dominantly prioritizes intramolecular cyclization by T4 ligase over intermolecular polymerization: an exclusive methodology for producing ssDNA rings.

When oligonucleotide bearing a hairpin near either its 3'- or 5'-end was treated with T4 DNA ligase, the intramolecular cyclization dominantly proceeded and its monomeric cyclic ring was obtained in extremely high selectivity. The selectivity was hardly dependent on the concentration of the oligonucleotide, and thus it could be added in one portion to the mixture at the beginning of the reaction. Without the hairpin, however, the formation of polymeric byproducts was dominant under the same conditions. Hairpin-bearing oligonucleotides primarily take the folded form, and the enzymatically reactive species (its open form) is minimal. As the result, the intermolecular reactions are efficiently suppressed due to both thermodynamic and kinetic factors. The 'terminal hairpin strategy' was applicable to large-scale preparation of a variety of DNA rings. The combination of this methodology with 'diluted buffer strategy', developed previously, is still more effective for the purpose. When large amount of l-DNA bearing a terminal hairpin (e.g. 40 µM) was treated in a diluted ligase buffer (0.1× buffer) with T4 DNA ligase, the DNA ring was prepared in 100% selectivity. Even at [l-DNA]0 = 100 µM in 0.1× buffer, the DNA ring was also obtained in pure form, simply by removing tiny quantity of linear byproducts by Exonuclease I.

Topologically Constrained Formation of Stable Z-DNA from Normal Sequence under Physiological Conditions.

Z-DNA, a left-handed duplex, has been shown to form in vivo and regulate expression of the corresponding gene. However, its biological roles have not been satisfactorily understood, mainly because Z-DNA is easily converted to the thermodynamically favorable B-DNA. Here we present a new idea to form stable Z-DNA under normal physiological conditions and achieve detailed analysis on its fundamental features. Simply by mixing two complementary minicircles of single-stranded DNA with no chemical modification, the hybridization spontaneously induces topological constraint which twines one-half of the double-stranded DNA into stable Z-DNA. The formation of Z-conformation with high stability has been proved by using circular dichroism spectroscopy, Z-DNA-specific antibody binding assay, nuclease digestion, etc. Even at a concentration of MgCl2 as low as 0.5 mM, Z-DNA was successfully obtained, avoiding the use of high salt conditions, limited sequences, ancillary additives, or chemical modifications, criteria which have hampered Z-DNA research. The resultant Z-DNA has the potential to be used as a canonical standard sample in Z-DNA research. By using this approach, further developments of Z-DNA science and its applications become highly promising.

The staining efficiency of cyanine dyes for single-stranded DNA is enormously dependent on nucleotide composition.

The staining of nucleic acids with fluorescent dyes is one of the most fundamental technologies in relevant areas of science. For reliable and quantitative analysis, the staining efficiency of the dyes should not be very dependent on the sequences of the specimens. However, this assumption has not necessarily been confirmed by experimental results, especially in the staining of ssDNA (and RNA). In this study, we found that both SYBR Green II and SYBR Gold did not stain either homopyrimidines or ssDNA composed of only adenine (A) and cytosine (C). However, these two dyes emit strong fluorescence when the ssDNA contains both guanine (G) and C (and/or both A and thymine (T)) and form potential Watson-Crick base pairs. Interestingly, SYBR Gold, but not SYBR Green II, strongly stains ssDNA consisting of G and A (or G and T). Additionally, we found that the secondary structure of ssDNA may play an important role in DNA staining. To obtain reliable results for practical applications, sufficient care must be paid to the composition and sequence of ssDNA.

Isothermal double-cycle catalytic system using DNAzyme and RNase H for the highly selective one-pot detection of oligonucleotides.

With the use of a double-cycle system involving two catalytic reactions by RNase H and DNAzyme, the signal of oligoDNAs has been specifically amplified in an isothermal mode. The precursor of DNAzyme was introduced to the system as a ring-structured and inactivated form, which involves the 6-nt RNA portion being complementary to target oligoDNA. In the presence of target oligoDNA, the RNA portion forms a DNA/RNA hetero-duplex and is cut by RNase H. This scission converts the precursor to catalytically active DNAzyme, which in turn disconnects the molecular beacon to produce the amplified signal. Because the covalent bonds were disconnected to provide discrete structural changes in both cycles, high sensitivity and specificity are obtained, indicating the strong potential of this double catalytic cycle method for versatile applications.

Highly efficient preparation of single-stranded DNA rings by T4 ligase at abnormally low Mg(II) concentration.

Preparation of large amount of single-stranded circular DNA in high selectivity is crucial for further developments of nanotechnology and other DNA sciences. Herein, a simple but practically useful methodology to prepare DNA rings has been presented. One of the essential factors is to use highly diluted T4 ligase buffer for ligase reactions. This strategy is based on our unexpected finding that, in diluted T4 buffers, intermolecular polymerization of DNA fragments is greatly suppressed with respect to their intramolecular cyclization. This promotion of cyclization is attributable to abnormally low concentration of Mg2+ ion (0.5-1.0 mM) but not ATP in the media for T4 ligase reactions. The second essential factor is to add DNA substrate intermittently to the mixture and maintain its temporal concentration low. By combining these two factors, single-stranded DNA rings of various sizes (31-74 nt) were obtained in high selectivity (89 mol% for 66-nt DNA) and in satisfactorily high productivity (∼0.2 mg/ml). A linear 72-nt DNA was converted to the corresponding DNA ring in nearly 100% selectivity. The superiority of this new method was further substantiated by the fact that small-sized DNA rings (31-42 nt), which were otherwise hardly obtainable, were successfully prepared in reasonable yields.

Colorimetric determination of mercury(II) ion based on DNA-assisted amalgamation: a comparison study on gold, silver and Ag@Au Nanoplates.

Inspired by the increasing use of plasmonic gold and silver nanoplates as probes for diverse analytes, the research community often questions which metal nanoplates should be chosen for a given application. A comparative study was performed on the performance and physicochemical properties of three types of metal nanoplates for use in plasmonic detection of Hg(II) ion. Specifically, gold, silver and Ag@Au nanoplates were studied. The established amalgamation method integrated into a detection scheme using nanoplates affords a unique yet straightforward signaling and extraction route for selective recognition of Hg(II) ion. Upon transformation of Hg(II) ion to metallic mercury, nanoplate amalgamation takes place instantly. This reshapes both the morphology and the optical characteristics of nanoplates. It is found that gold and Ag@Au nanoplates enable highly selective quantitation of Hg(II) ion by using a DNA oligomer consisting of poly-deoxycytidine (poly(C)) as a masking agent against Ag(I) ion. The silver nanoplates, in turn, display the best sensitivity owing to the chemical instability. The induced surface plasmonic shifts (of up to 250 nm and color changes from red to green) allows for determination of Hg(II) over a wide range and with a limit of detection of ~10 nM. It is recommended that the gold and Ag@Au nanoplates are used in relatively complex systems, while silver nanoplates are suited for simple matrices. Graphic abstract The amalgamation process integrated with metal (e.g., Au, Ag and Ag@Au) nanoplates affords plasmonic detection of Hg(II) ion with the aid of a poly(c) DNA sequence as the masking agent for Ag(I) ion.

One-Pot Isolation of a Desired Human Genome Fragment by Using a Biotinylated pcPNA/S1 Nuclease Combination.

Scission of the human genome at predetermined sites and isolation of a particular fragment are of great interest for the analysis of lesion/modification sites, in proteomics, and for gene therapy. However, methods for human genome scission and specific fragment isolation are limited. Here, we report a novel one-pot method for the site-specific scission of DNA by using a biotinylated pcPNA/S1 nuclease combination and isolation of a desired fragment by streptavidin-coated magnetic beads. The proof of concept was initially demonstrated for the clipping of plasmid DNA and isolation of the required fragment. Our method was then successfully applied for the isolation of a fragment from the cell-derived human genome.

Two-Holder Strategy for Efficient and Selective Synthesis of Lk 1 ssDNA Catenane.

DNA catenanes are characterized by their flexible and dynamic motions and have been regarded as one of the key players in sophisticated DNA-based molecular machines. There, the linking number (Lk) between adjacent interlocked rings is one of the most critical factors, since it governs the feasibility of dynamic motions. However, there has been no established way to synthesize catenanes in which Lk is controlled to a predetermined value. This paper reports a new methodology to selectively synthesize Lk 1 catenanes composed of single-stranded DNA rings, in which these rings can most freely rotate each other due to minimal inter-ring interactions. To the mixture for the synthesis, two holder strands (oligonucleotides of 18⁻46 nt) were added, and the structure of the quasi-catenane intermediate was interlocked through Watson⁻Crick base pairings into a favorable conformation for Lk 1 catenation. The length of the complementary part between the two quasi-rings was kept at 10 bp or shorter. Under these steric constraints, two quasi-rings were cyclized with the use of T4 DNA ligase. By this simple procedure, the formation of undesired topoisomers (Lk ≥ 2) was almost completely inhibited, and Lk 1 catenane was selectively prepared in high yield up to 70 mole%. These Lk 1 catenanes have high potentials as dynamic parts for versatile DNA architectures.

Applications of PNA-Based Artificial Restriction DNA Cutters.

More than ten years ago, artificial restriction DNA cutters were developed by combining two pseudo-complementary peptide nucleic acid (pcPNA) strands with either Ce(IV)/EDTA or S1 nuclease. They have remarkably high site-specificity and can cut only one predetermined site in the human genome. In this article, recent progress of these man-made tools have been reviewed. By cutting the human genome site-selectively, desired fragments can be clipped from either the termini of chromosomes (telomeres) or from the middle of genome. These fragments should provide important information on the biological functions of complicated genome system. DNA/RNA hybrid duplexes, which are formed in living cells, are also site-selectively hydrolyzed by these cutters. In order to further facilitate the applications of the artificial DNA cutters, various chemical modifications have been attempted. One of the most important successes is preparation of PNA derivatives which can form double-duplex invasion complex even under high salt conditions. This is important for in vivo applications, since the inside of living cells is abundant of metal ions. Furthermore, site-selective DNA cutters which require only one PNA strand, in place of a pair of pcPNA strands, are developed. This progress has opened a way to new fields of PNA-based biochemistry and biotechnology.

Click conjugation of a binuclear terbium(III) complex for real-time detection of tyrosine phosphorylation.

Phosphorylation of proteins is closely associated with various diseases, and, therefore, its detection is vitally important in molecular biology and drug discovery. Previously, we developed a binuclear Tb(III) complex, which emits notable luminescence only in the presence of phosphotyrosine. In this study, we conjugated a newly synthesized binuclear Tb(III) complex to substrate peptides by using click chemistry. Using these conjugates, we were able to detect tyrosine phosphorylation in real time. These conjugates were superior to nonconjugated Tb(III) complexes for the detection of tyrosine phosphorylation, especially when the substrate peptides used were positively charged. Luminescence intensity upon phosphorylation was enhanced 10-fold, making the luminescence intensity of this system one of the largest among lanthanide luminescence-based systems. We also determined Michaelis-Menten parameters for the phosphorylation of various kinase/peptide combinations and quantitatively analyzed the effects of mutations in the peptide substrates. Furthermore, we successfully monitored the inhibition of enzymatic phosphorylation by inhibitors in real time. Advantageously, this system detects only the phosphorylation of tyrosine (phosphorylated serine and threonine are virtually silent) and is applicable to versatile peptide substrates. Our study thus demonstrates the applicability of this system for the analysis of kinase activity, which could lead to drug discovery.

Promotion of double-duplex invasion of peptide nucleic acids through conjugation with nuclear localization signal peptide.

Pseudo-complementary peptide nucleic acid (pcPNA), as one of the most widely used synthetic DNA analogues, invades double-stranded DNA according to Watson-Crick rules to form invasion complexes. This unique mode of DNA recognition induces structural changes at the invasion site and can be used for a range of applications. In this paper, pcPNA is conjugated with a nuclear localization signal (NLS) peptide, and its invading activity is notably promoted both thermodynamically and kinetically. Thus, the double-duplex invasion complex is formed promptly at low pcPNA concentrations under high salt conditions, where the invasion otherwise never occurs. Furthermore, NLS-modified pcPNA is successfully employed for site-selective DNA scission, and the targeted DNA is selectively cleaved under conditions that are not conducive for DNA cutters using unmodified pcPNAs. This strategy of pcPNA modification is expected to be advantageous and promising for a range of in vitro and in vivo applications.

Image monitoring of pharmaceutical blending processes and the determination of an end point by using a portable near-infrared imaging device based on a polychromator-type near-infrared spectrometer with a high-speed and high-resolution photo diode array detector.

In the present study we have developed a new version (ND-NIRs) of a polychromator-type near-infrared (NIR) spectrometer with a high-resolution photo diode array detector, which we built before (D-NIRs). The new version has four 5 W halogen lamps compared with the three lamps for the older version. The new version also has a condenser lens with a shorter focal point length. The increase in the number of the lamps and the shortening of the focal point of the condenser lens realize high signal-to-noise ratio and high-speed NIR imaging measurement. By using the ND-NIRs we carried out the in-line monitoring of pharmaceutical blending and determined an end point of the blending process. Moreover, to determinate a more accurate end point, a NIR image of the blending sample was acquired by means of a portable NIR imaging device based on ND-NIRs. The imaging result has demonstrated that the mixing time of 8 min is enough for homogeneous mixing. In this way the present study has demonstrated that ND-NIRs and the imaging system based on a ND-NIRs hold considerable promise for process analysis.

Finding a human telomere DNA-RNA hybrid G-quadruplex formed by human telomeric 6-mer RNA and 16-mer DNA using click chemistry: a protective structure for telomere end.

Telomeric repeat-containing RNA is a non-coding RNA molecule newly found in mammalian cells. The telomere RNA has been found to localize to the telomere DNA, but how the newly discovered RNA molecule interacts with telomere DNA is less known. In this study, using the click chemistry we successfully found that a 6-mer human telomere RNA and 16-mer human telomere DNA sequence can form a DNA-RNA hybrid type G-quadruplex structure. Detection of the click-reaction products directly probes DNA-RNA G-quadruplex structures in a complicated solution, whereas traditional methods such as NMR and crystallography may not be suitable. Importantly, we found that formation of DNA-RNA G-quadruplex induced an exonuclease resistance for telomere DNA, indicating that such structures might be important for protecting telomeric DNA from enzyme digestion to avoid telomere DNA shortening. These results provide the direct evidence for formation of DNA-RNA hybrid G-quadruplex structure by human telomere DNA and RNA sequence, suggesting DNA-RNA hybrid G-quadruplex structure associated between telomere DNA and RNA may respond to chromosome end protection and/or present a valuable target for drug design.