Formation of an instantaneous nick for highly efficient adenylation of oligonucleotides by ligase without subsequent jointing.

By forming a nick at the adenylation site instantaneously, nucleic acids are efficiently adenylated by T4 DNA ligase. The subsequent ligation is successfully suppressed in terms of rapid conversion of the instantaneous nick to a more stable gap. It is helpful to understand enzymatic ligation dynamics, and the adenylated products can be used for various practical applications.

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.

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.

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.

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.

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.

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.

Effect of sulfated polysaccharides on the digestion of DNA by pepsin under simulated gastric juice in vitro.

The effect of sulfated polysaccharides on the digestion of dietary DNA by pepsin was studied using in vitro simulated gastric juice. The results showed that fucoidan (FUC), dextran sulfate (DS) and chondroitin sulfate (CS) could inhibit the digestion of DNA in a dose-dependent manner. Polysaccharides with high sulfate group content have stronger inhibition ability. Fluorescence spectroscopy results showed that polysaccharides could bind to pepsin, and transmission electron microscopy (TEM) confirmed that polysaccharides can interact with DNA, which not only is the main reason that polysaccharides inhibit the digestion of DNA by pepsin but also causes the digestion of DNA by DNase II to be inhibited. The finding suggests that the digestion of DNA should be reevaluated when eating foods rich in sulfated polysaccharides. This study enriched the known pharmacological properties of sulfated polysaccharides as pepsin inhibitors and provided inspiration for the use of sulfated polysaccharides as oligonucleotide drug delivery carriers.

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.

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.

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.

Efficient Preparation of Large-Sized Rings of Single-Stranded DNA through One-Pot Ligation of Multiple Fragments.

Circular single-stranded DNA (c-ssDNA) has significant applications in DNA detection, the development of nucleic acid medicine, and DNA nanotechnology because it shows highly unique features in mobility, dynamics, and topology. However, in most cases, the efficiency of c-ssDNA preparation is very low because polymeric byproducts are easily formed due to intermolecular reaction. Herein, we report a one-pot ligation method to efficiently prepare large c-ssDNA. By ligating several short fragments of linear single-stranded DNA (l-ssDNA) in one-pot by using T4 DNA ligase, longer l-ssDNAs intermediates are formed and then rapidly consumed by the cyclization. Since the intramolecular cyclization reaction is much faster than intermolecular polymerization, the formation of polymeric products is suppressed and the dominance of intramolecular cyclization is promoted. With this simple approach, large-sized single-stranded c-ssDNAs (e.g., 200-nt) were successfully synthesized in high selectivity and yield.

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.

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.

Artificial Restriction DNA Cutter Using Nuclease S1 for Site-Selective Scission of Genomic DNA.

By combining a pair of pseudo-complementary peptide nucleic acids (pcPNAs) with S1 nuclease, a novel tool to cut DNA at a predetermined site can be obtained. Complementary pcPNAs invade the DNA duplex and base pair to each strand of a target site, creating single-stranded regions that are cleaved by S1 nuclease. The scission site can be freely modulated by the design of pcPNAs. This method can be used to cleave a single site in the human genome. This protocol presents experimental details for site-selective scission using this versatile new tool. © 2019 by John Wiley & Sons, Inc.

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.

Effects of secondary structures of DNA templates on the quantification of qPCR.

In the current design of quantitative polymerase chain reaction (qPCR) systems, the sequences of primers are the primary concerns. The secondary structures of DNA templates have not been much considered, although they should be also critically important. In this paper, various hairpins with different stem lengths and loop sizes are placed near primer-binding sites, and their effects on the amplification efficiency of qPCR are systematically investigated. When a hairpin is formed either in the inside of the amplicon or in its outside, the amplification is notably suppressed. The magnitudes of suppression increase with the increase in stem length and the decrease in loop size, and are especially significant for the hairpins formed inside the amplicon. With very long stems (e.g., 20-bp), the effect is still more drastic, and no targeted amplification products are formed. On the basis of melting temperature (Tm) measurements, these suppression effects of hairpins have been mostly ascribed to competitive inhibition of primer binding to the template. It has been concluded that, in order to design precise and reliable qPCR systems, at least 60-bp sequences around primer-binding sites, both inside and outside the amplicons, must be analyzed to confirm that stable secondary structures are not formed in the vicinity of primer-binding sites. Communicated by Ramaswamy H. Sarma.

RNA ligation of very small pseudo nick structures by T4 RNA ligase 2, leading to efficient production of versatile RNA rings.

T4 RNA ligase 2 catalyses two types of reactions: (i) sealing of a nick structure in double-stranded RNA and (ii) connection of two single-stranded RNA strands. In order to obtain comprehensive views on these two types of reactions and widen the application scope of this RNA ligase, we here systematically analysed the connection of single-stranded RNA strands having different secondary structures. It has been found that the ligation is enormously promoted when a stem of only 4-bp or longer is formed in the 3'-OH side of the joining site. Additional placement of a stem in the 5'-phosphate side further facilitates the ligation. In contrast, perturbation of the stem structures in RNA substrates suppresses the ligation. These results indicate that ligation of two single-stranded RNA strands by T4 RNA ligase 2 is greatly promoted by forming a "nick-like intermediate". Even the unstable intermediate, formed only temporarily in the solution, is sufficiently effective. By designing the synthetic systems in terms of this finding, short single-stranded RNA rings of versatile sizes, which are otherwise hard to be obtained, are efficiently prepared in high selectivity and yield.

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.