Direct dsRNA preparation by promoter-free RCT and RNase H cleavage using one circular dsDNA template with a mismatched bubble.

Double-stranded RNA (dsRNA) has aroused widespread interest due to its effects on immunity and applications based on RNAi. However, the in vitro preparation of dsRNA is costly and laborious. In this study, we have developed a novel and interesting method designated as pfRCT (promoter-free rolling-circle transcription) for direct, facile, and efficient dsRNA preparation. This method generates equal amounts of sense and antisense strands simultaneously from a single circular dsDNA template. To initiate transcription by T7 RNA polymerase without directional preference, a 9-15-bp bubble (mismatched duplex with strong sequence symmetry) is introduced into the template. During RCT, all the necessary reagents, including the template, NTPs, RNA polymerase, RNase H, and Helpers, are present in one pot; and the just-transcribed RNA is immediately truncated by RNase H to monomers with the desired size. The ends of the dsRNA product can also be simply sealed by T4 RNA ligase 1 after pfRCT. This new approach is expected to promote the applications of dsRNA.

Nonalternating purine pyrimidine sequences can form stable left-handed DNA duplex by strong topological constraint.

In vivo, left-handed DNA duplex (usually refers to Z-DNA) is mainly formed in the region of DNA with alternating purine pyrimidine (APP) sequence and plays significant biological roles. It is well known that d(CG)n sequence can form Z-DNA most easily under negative supercoil conditions, but its essence has not been well clarified. The study on sequence dependence of Z-DNA stability is very difficult without modification or inducers. Here, by the strong topological constraint caused by hybridization of two complementary short circular ssDNAs, left-handed duplex part was generated for various sequences, and their characteristics were investigated by using gel-shift after binding to specific proteins, CD and Tm analysis, and restriction enzyme cleavage. Under the strong topological constraint, non-APP sequences can also form left-handed DNA duplex as stable as that of APP sequences. As compared with non-APP sequences, the thermal stability difference for APP sequences between Z-form and B-form is smaller, which may be the reason that Z-DNA forms preferentially for APP ones. This result can help us to understand why nature selected APP sequences to regulate gene expression by transient Z-DNA formation, as well as why polymer with chirality can usually form both duplexes with left- or right-handed helix.

Unexpected Mechanism and Inhibition Effect for Nonspecific Amplification Involving Dynamic Binding of Primers with Background DNA.

Nonspecific amplification is a serious issue in DNA detection as it can lead to false-positive results and reduce specificity. It is very important to well understand its mechanism through sequencing nonspecific products. Here, an approach is developed using a nanopore sequencing technique after acquiring the long repetitive sequence of DNA products from nonspecific amplification. Based on the sequencing results, a new mechanism of nonspecific amplification designated as dynamic mismatched primer binding (DMPB) with the background DNA (bgDNA) is proposed. Unexpectedly, our findings show that the primers (∼20 nt) can bind to bgDNA for primer extension when only 6-11 fully matched (9-14 mismatched) base pairs are formed. After the single-stranded DNAs (ssDNAs) attached to the first primer are produced, more interestingly, with the aid of DNA polymerase, the other primer can bind to these ssDNAs in the case that the fully matched base pairs formed between them are even shorter than 6 bp. As a result, perfect "seeds" for polymerase chain reaction with information on both primers are produced so that exponential nonspecific amplification can occur. The DMPB mechanism can explain nonspecific amplification in other approaches as well. Finally, a mini-hairpin DNA is used to effectively inhibit nonspecific amplification by preventing the formation of an unexpected primer-bgDNA complex.

Effects of ribonucleotide supplementation in modulating the growth of probiotic Bacillus subtilis and the synergistic benefits for improving the health performance of Asian seabass (Lates calcarifer).

In aquaculture, due to the requirements for high-density culture, the diseases caused by bacterial pathogens have become a serious issue. To solve this problem, we performed synbiotic application of RNA and Bacillus subtilis as a sustainable and eco-friendly approach to improve the health and immunity of Asian seabass (Lates calcarifer) during cultivation without using any harmful antibiotics or chemicals. Among various forms of nucleic acids, such as mononucleotides and DNA, RNA was found to be most effective in promoting the growth performance of probiotic B. subtilis in all the tested minimal medium conditions. Accordingly, we used the synbiotic combination of B. subtilis and RNA for Asian seabass cultivation. After feed supplementation for fourteen days, the fish that received the combination treatment exhibited a significant increase in innate cellular and humoral immune parameters, including phagocytic activity, phagocytic index, respiratory burst, serum lysozyme and bactericidal activities, as well as upregulated expression of immune-related genes, including HEPC1, A2M, C3, CC, CLEC, LYS, HSP70, and HSP90. Furthermore, significant increases were observed in the ileal villus height and goblet cell numbers in the intestinal villi in all fish treatment groups. The combination treatment did not cause histopathological abnormalities in the intestine and liver, suggesting that the synbiotic treatment is safe for use in fish. The treated Asian seabass also exhibited a significantly increased survival rate after Aeromonas hydrophila challenge. These results indicate that the synbiotic mixture of B. subtilis and RNA can be considered a beneficial feed additive and immunostimulant for Asian seabass cultivation.

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.

Construction of ssDNA-Attached LR-Chimera Involving Z-DNA for ZBP1 Binding Analysis.

The binding of proteins to Z-DNA is hard to analyze, especially for short non-modified DNA, because it is easily transferred to B-DNA. Here, by the hybridization of a larger circular single-stranded DNA (ssDNA) with a smaller one, an LR-chimera (involving a left-handed part and a right-handed one) with an ssDNA loop is produced. The circular ssDNAs are prepared by the hybridization of two ssDNA fragments to form two nicks, followed by nick sealing with T4 DNA ligase. No splint (a scaffold DNA for circularizing ssDNA) is required, and no polymeric byproducts are produced. The ssDNA loop on the LR-chimera can be used to attach it with other molecules by hybridization with another ssDNA. The gel shift binding assay with Z-DNA specific binding antibody (Z22) or Z-DNA binding protein 1 (ZBP1) shows that stable Z-DNA can form under physiological ionic conditions even when the extra ssDNA part is present. Concretely, a 5'-terminal biotin-modified DNA oligonucleotide complementary to the ssDNA loop on the LR-chimera is used to attach it on the surface of a biosensor inlaid with streptavidin molecules, and the binding constant of ZBP1 with Z-DNA is analyzed by BLI (bio-layer interferometry). This approach is convenient for quantitatively analyzing the binding dynamics of Z-DNA with other molecules.

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.

Plasmon switching of gold nanoparticles through thermo-responsive terminal breathing of surface-grafted DNA in hydrated ionic liquids.

Self-assembly performed in ionic liquids (ILs) as a unique solvent promises distinct functions and applications in sensors, therapeutics, and optoelectronic devices due to the rich interactions between nanoparticle building blocks and ILs. However, the general consideration that common nanoparticles are readily destabilized by counterions in an IL has largely prevented researchers from investigating controlled nanoparticle assembly in IL-based systems. This study explores the assembling behaviour of double-stranded (ds) DNA-functionalized gold nanoparticles (dsDNA-AuNPs) in hydrated ionic liquids. The DNA base pair stacking assembly of dsDNA-AuNPs occurs at a low IL concentration (<5%). However, a moderate ionic liquid concentration (5-40%) can de-hybridize dsDNA and leaves single-stranded (ss) DNA stabilizing the AuNPs. In concentrated ionic liquids (>40%), interestingly, the higher ionic strength leads to the assembly of DNA-AuNPs. The triphasic assembly trend is also generally observed regardless of the type of IL. By down-regulation of DNA's melting temperature with the IL, the assembly of DNA-AuNPs affords robust response to a lower temperature range, promising applications in plasmonic devices and range-tunable temperature sensors.

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.

Accelerated non-crosslinking assembly of DNA-functionalized nanoparticles in alcoholic solvents: for application in the identification of clear liquors.

Colorimetric detection of various target molecules in aqueous solutions based on the non-crosslinking assembly of DNA-functionalized Au nanoparticles (DNA-AuNPs) has been well established in recent years. The extension of DNA-AuNPs to other solvents remains much less explored, despite the practical importance of detection in non-aqueous solutions, such as those containing an organic ingredient that is required or not removable in many contexts. However, the general consideration that DNA is easily denatured and precipitated in organic solvents has been hampering the use of DNA-AuNPs in low polar solvents. Herein, we report a more rapid non-crosslinking assembly of double-stranded (ds) DNA-AuNPs in alcoholic solvents than in aqueous solvents. When the concentration of ethanol in the disperse medium is increased from 0% to 20% (v/v), the rate of non-crosslinking assembly is distinctly increased by a factor of 5-6, whereas the rate is sharply decreased when the ethanol concentration is further increased to 40%. This biphasic kinetics trend could be attributed to the competitive balance between the enhanced intermolecular attraction between dsDNAs and the increased propensity for melting of dsDNA. Rapid naked-eye identification of clear liquors that are encoded by oligonucleotide additives has also been demonstrated by using the alcoholic non-crosslinking assembly of dsDNA-AuNPs as a proof-of-concept.

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.

Engineering Aptamer with Enhanced Affinity by Triple Helix-Based Terminal Fixation.

The affinity of aptamers relies on their adaptive folding, but the excessive flexibility of the aptamer backbone usually hampers the folding process. Thus, there is an urgent need to engineer aptamers with more stable and defined structures. Herein, we report a postselection strategy for stabilizing aptamer structures, by fixing both termini of the aptamer with a length-optimized triple helix structure. An anti-lysozyme aptamer was engineered in this way, and its affinity was enhanced by almost 10-fold. An electrochemical aptasensor was designed based on this engineered aptamer, assisted by a DNA tetrahedron as a spacer to orient the aptamer. The aptasensor achieved a 180-fold lower limit of detection than that achieved by the aptasensor without termini-fixed aptamer and exhibited high sensitivity and selectivity toward lysozyme in real red wine samples. This work sheds light on engineering aptamers to achieve enhanced affinity and on the application of aptasensors in complex matrices.

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.

Chemically Fueled Plasmon Switching of Gold Nanorods by Single-Base Pairing of Surface-Grafted DNA.

The interactions between metal ions and biomolecules are crucial to various bioprocesses. Development of plasmon switching nanodevices that exploit these molecular interactions is of fundamental and technological interest. Here, we show plasmon switching based on rapid aggregation/dispersion of double-stranded DNA-modified gold nanorods (dsDNA-AuNRs) that exhibit colloidal behaviors depending on pairing/unpairing of the terminal bases. The dsDNA-AuNRs bearing a thymine-thymine (T-T) mismatch at the penultimate position undergo spontaneous non-cross-linking aggregation in the presence of Hg2+ due to T-Hg-T base pairing. Inversely, the subsequent addition of cysteine (Cys) gives rise to the removal of Hg2+ from the T-Hg-T base pair to reproduce the T-T mismatch, resulting in stable dispersion of the dsDNA-AuNRs. The chemical-responsive plasmon switch allows for the rapid and repeatable cycles at room temperature. The validity of the present method is further exemplified by developing another plasmon switch fueled by Ag+ and Cys by installing the Ag+-binding DNA sequence in the dsDNA-AuNR.

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.