Kinetics of Strand Displacement Reaction with Acyclic Artificial Nucleic Acids.

Toehold-mediated strand displacement (TMSD) reaction, one of the DNA nanotechnologies, has great potential as s biological programmable platform in the cellular environment. Various artificial nucleic acids have been developed to improve stability and affinity for biological applications. However, the lack of understanding of the kinetics of TMSD reaction among artificial nucleic acids has limited their applications. We herein systematically characterized the kinetics of TMSD reactions with acyclic xeno nucleic acids (XNAs): serinol nucleic acid (SNA), acyclic D-threoninol nucleic acid (D-aTNA), and acyclic L-threoninol nucleic acid (L-aTNA). We found that the strand displacement reactions by D-aTNA and by L-aTNA were highly dependent on temperature. D-aTNA and L-aTNA systems were orthogonal to each other, and chirality of the input can be switched by using SNA as an interface. We also applied TMSD reactions of XNAs to a seesaw gate amplification system which utilizes the orthogonality. This work will contribute to the developments of thermoresponsive and bioorthogonal nucleic acid circuits.

A Light-powered single-stranded DNA molecular motor with colour-selective single-step control.

To-down control of small motion is possible through top-down controlled molecular motors in replacement of larger actuators like MEMS or NEMS (micro- or nano-electromechanical systems) in the current precision technology. Improving top-down control of molecular motors to every single step is desirable for this purpose, and also for synchronization of motor actions for amplified effects. Here we report a designed single-stranded DNA molecular motor powered by alternated ultraviolet and visible light for processive track-walking, with the two light colours each locking the motor in a full directional step to allow saturated driving but no overstepping. This novel nano-optomechanical driving mechanism pushes the top-down control of molecular motors down to every single step, thus providing a key technical capability to advance the molecular motor-based precision technology and also motor synchronization for amplified effects.

Photosensitization of TiO2 electrodes immobilized with chiral plasmonic Au nanocolloids by circularly polarized light irradiation.

Photosensitization of semiconductors by excitation of chiral plasmonic metallic nanostructures has attracted much attention, not only for the analysis and detection of circularly polarized light but also for its potential applications in chiral photosynthesis. Although there have been reports on the detection of semiconductor-sensitized current in chiral nanostructures precisely fabricated by physical vapor deposition and/or lithography techniques, there have been no studies using plasmonic metal nanocolloids synthesized by chemical processes. In this study, we report the establishment of a fabrication method for large-area chiral photoelectrodes and the semiconductor photosensitization phenomenon realized using chiral plasmonic nanoparticles. Chiral plasmonic Au nanoparticles prepared by previously reported colloidal methods were immobilized onto a TiO2 thin film electrode by electrophoresis. When TiO2 electrodes loaded with chiral Au nanoparticles synthesized using L-cysteine were irradiated with circularly polarized light, left circularly polarized light irradiation at a wavelength of 500-600 nm generated a larger anodic photocurrent than right circularly polarized light irradiation at the same wavelength. This trend was reversed for TiO2 electrodes immobilized with colloidal Au nanoparticles synthesized with D-cysteine. From these results, we conclude that the efficiency of photocurrent generation by chiral plasmon excitation can be controlled by the polarization direction of the incident light.

DNA Reaction System That Acquires Classical Conditioning.

Biochemical reaction networks can exhibit plastic adaptation to alter their functions in response to environmental changes. This capability is derived from the structure and dynamics of the reaction networks and the functionality of the biomolecule. This plastic adaptation in biochemical reaction systems is essentially related to memory and learning capabilities, which have been studied in DNA computing applications for the past decade. However, designing DNA reaction systems with memory and learning capabilities using the dynamic properties of biochemical reactions remains challenging. In this study, we propose a basic DNA reaction system design that acquires classical conditioning, a phenomenon underlying memory and learning, as a typical learning task. Our design is based on a simple mechanism of five DNA strand displacement reactions and two degradative reactions. The proposed DNA circuit can acquire or lose a new function under specific conditions, depending on the input history formed by repetitive stimuli, by exploiting the dynamic properties of biochemical reactions induced by different input timings.

Site-Selective Photo-Crosslinking of Stilbene Pairs in a DNA Duplex Mediated by Ruthenium Photocatalyst.

We herein report a method for site-selective photo-crosslinking of a DNA duplex. A stilbene pair was introduced into a DNA duplex and a ruthenium complex was conjugated with a triplex-forming oligonucleotide. We demonstrated that [2+2] photocycloaddition of the stilbene pair occurred upon irradiation with visible light when the ruthenium complex was in close proximity due to triplex formation. No reaction occurred when the ruthenium complex was not in proximity to the stilbene pair. The wavelength of visible light used was of lower energy than the wavelength of UV light necessary for direct excitation of stilbene. Quantum chemical calculation indicated that ruthenium complex catalyzed the photocycloaddition via triplet-triplet energy transfer. Site selectivity of this photo-crosslinking system was evaluated using a DNA duplex bearing two stilbene pairs as a substrate; we showed that the site of crosslinking was precisely regulated by the sequence of the oligonucleotide linked to the ruthenium complex. Since this method does not require orthogonal photoresponsive molecules, it will be useful in construction of complex photoresponsive DNA circuits, nanodevices and biological tools.

Unexpectedly stable homopurine parallel triplex of SNA:RNA*SNA and L-aTNA:RNA*L-aTNA.

Homopurine strands are known to form antiparallel triplexes stabilized by G*G and A*A Hoogsteen pairs, which have two hydrogen bonds. But there has been no report on the parallel triplex formation of homopurine involving both adenosine and guanosine to the duplex. In this paper, we first report parallel triplex formation between a homopurine serinol nucleic acid (SNA) strand and an RNA/SNA duplex. Melting profiles revealed that the parallel SNA:RNA*SNA triplex was remarkably stable, even though the A*A pair has a single hydrogen bond. An L-acyclic threoninol nucleic acid (L-aTNA) homopurine strand also formed a stable parallel triplex with an L-aTNA/RNA duplex.

Illuminating miRNA Inhibition: Visualizing the Interaction between Anti-miRNA Oligonucleotide and Target miRNA Using FRET.

Anti-miRNA oligonucleotides (anti-miRs) effectively and specifically inhibit the function of individual miRNAs and have the potential to serve as a novel class of nucleic acid therapeutic. However, the details of the mechanisms of anti-miRs in cells have not yet been clarified sufficiently. In particular, the localization of the complexes of anti-miRs and target miRNA in cells remains unclear. We previously developed anti-miRs composed of serinol nucleic acid (SNA) that very effectively inhibited miRNA-mediated silencing activity. Here we describe an imaging system based on the fluorescence resonance energy transfer (FRET) designed by miRNAs labeled with fluorophore-quencher pairs and an SNA-based anti-miR labeled with an acceptor dye. We discovered that the anti-miR hybridizes with the miRNA in the miRNA-induced silencing complex (miRISC), which is the active complex formed by miRNA and Ago2 in cells within P-bodies. Based on FRET ratio analysis, we hypothesize that the complex formed by the anti-miR and the miRNA in P-bodies is dynamic, with anti-miR complexing the miRISC, followed by miRNA release and degradation. Our findings provide valuable insights into the mechanism of action of anti-miRs and enable further studies of miRNA-targeted therapeutics.

Rapid Chemical Ligation of DNA and Acyclic Threoninol Nucleic Acid (aTNA) for Effective Nonenzymatic Primer Extension.

Previously, nonenzymatic primer extension reaction of acyclic l-threoninol nucleic acid (L-aTNA) was achieved in the presence of N-cyanoimidazole (CNIm) and Mn2+; however, the reaction conditions were not optimized and a mechanistic insight was not sufficient. Herein, we report investigation of the kinetics and reaction mechanism of the chemical ligation of L-aTNA to L-aTNA and of DNA to DNA. We found that Cd2+, Ni2+, and Co2+ accelerated ligation of both L-aTNA and DNA and that the rate-determining step was activation of the phosphate group. The activation was enhanced by duplex formation between a phosphorylated L-aTNA fragment and template, resulting in unexpectedly more effective L-aTNA ligation than DNA ligation. Under optimized conditions, an 8-mer L-aTNA primer could be elongated by ligation to L-aTNA trimers to produce a 29-mer full-length oligomer with 60% yield within 2 h at 4 °C. This highly effective chemical ligation system will allow construction of artificial genomes, robust DNA nanostructures, and xeno nucleic acids for use in selection methods. Our findings also shed light on the possible pre-RNA world.

Orientational Control of Circularly Polarized Luminescence from Pyrene Clusters by Using a DNA Scaffold.

Invited for the cover of this issue is the group of Hiromu Kashida and Hiroyuki Asanuma at Nagoya University and co-workers. The image depicts the orientation dependence of circularly polarized luminescent. Read the full text of the article at 10.1002/chem.202300182.

Orientational Control of Circularly Polarized Luminescence from Pyrene Clusters by Using a DNA Scaffold.

We have investigated the chiroptical activities of pyrene clusters incorporated within a DNA duplex. Three pyrene derivatives were prepared on d-threoninol linkers to allow incorporation within a DNA strand. DNA scaffolds containing dimers, tetramers, and hexamers of the pyrene derivatives were prepared. The homodimers of 1- and of 4-pyrenecarboxylic acid, but not 2-pyrenecarboxylic acid, emitted intense circularly polarized luminescence signals. Although increasing the number of pyrene units weakened the signal, insertion of natural base pairs between two dimers enhanced its intensity. Interestingly, circularly polarized luminescence intensities varied non-monotonically depending on the number of intervening base pairs, thus indicating the importance of orientation between pyrene dimers. The results presented here could lead to the development of bright circularly polarized luminescence materials and probes.

Syntheses of Base-Labile Pseudo-Complementary SNA and l-aTNA Phosphoramidite Monomers.

We previously synthesized phosphoramidite monomers bearing Boc-protected 2,6-diaminopurine (D) and 2-methyl-4-methoxybenzyl-protected 2-thiouracil (sU) as building blocks for the preparation of pseudo-complementary serinol nucleic acids (SNAs). Since SNA is stable under acidic conditions, an acid-deprotection step could be inserted into the work-up. However, as the 4,4'-dimethoxytrityl group was concurrently removed at this step, purification of SNA by reversed-phase HPLC was difficult. Here, we report the syntheses of SNA and acyclic l-threoninol nucleic acid (l-aTNA) phosphoramidite monomers with bis(phenoxyacetyl)-protected D and 4-acetoxybenzyl-protected sU, both of which can be deprotected under mild basic conditions. Using these monomers, we prepared pseudo-complementary SNA and l-aTNA in high yield using conventional oligonucleotide synthesis protocols. These monomers can be used for large-scale syntheses of SNAs and l-aTNAs.

Methyl group configuration on acyclic threoninol nucleic acids (aTNAs) impacts supramolecular properties.

We have synthesized acyclic allo-threoninol nucleic acids (allo-aTNAs), artificial xeno-nucleic acids (XNAs) that are diastereomers of acyclic threoninol nucleic acids (aTNAs), and have investigated their supramolecular properties. The allo-aTNAs formed homo-duplexes in an antiparallel manner but with lower thermal stability than DNA, whereas aTNAs formed extremely stable homo-duplexes. The allo-aTNAs formed duplexes with complementary aTNAs and serinol nucleic acid (SNA). The affinities of L-allo-aTNA were the highest for L-aTNA and the lowest for D-aTNA, with SNA being intermediate. The affinities of D-allo-aTNA were the reverse. Circular dichroism measurements revealed that L- and D-allo-aTNAs had weak right-handed and left-handed helicities, respectively. The weak helicity of allo-aTNAs likely explains the poor chiral discrimination of these XNAs, which is in contrast to aTNAs that have strong helical orthogonality. Energy-minimized structures of L-allo-aTNA/RNA and L-allo-aTNA/L-allo-aTNA indicated that the methyl group on the allo-aTNA strand is unfavourable for duplex formation. In contrast, the methyl group on L-aTNA likely stabilizes the duplex structure via hydrophobic effects and van der Waals interactions. Thus, the configuration of the methyl group on the XNA scaffold had an unexpectedly large impact on the hybridization ability and structure.

Orthogonal Amplification Circuits Composed of Acyclic Nucleic Acids Enable RNA Detection.

Construction of complex DNA circuits is difficult due to unintended hybridization and degradation by enzymes under biological conditions. We herein report a hybridization chain reaction (HCR) circuit composed of left-handed acyclic d-threoninol nucleic acid (d-aTNA), which is orthogonal to right-handed DNA and RNA. Because of its high thermal stability, use of an aTNA hairpin with a short 7 base-pair stem ensured clear ON-OFF control of the HCR circuit. The aTNA circuit was stable against nucleases. A circuit based on right-handed acyclic l-threoninol nucleic acid (l-aTNA) was also designed, and high orthogonality between d- and l-aTNA HCRs was confirmed by activation of each aTNA HCR via a corresponding input strand. A dual OR logic gate was successfully established using serinol nucleic acid (SNA), which could initiate both d- and l-aTNA circuits. The d-aTNA HCR was used for an RNA-dependent signal amplification system via the SNA interface. The design resulted in 80% yield of the cascade reaction in 3000 s without a significant leak. This work represents the first example of use of heterochiral HCR circuits for detection of RNA molecules. The method has potential for direct visualization of RNA in vivo and the FISH method.

Renewable DNA Proportional-Integral Controller with Photoresponsive Molecules.

A molecular robot is an intelligent molecular system. A typical control problem of molecular robots is to maintain the concentration of a specific DNA strand at the desired level, which is typically attained by a molecular feedback control mechanism. A molecular feedback system can be constructed in a bottom-up method by transforming a nonlinear chemical reaction system into a pseudo-linear system. This method enables the implementation of a molecular proportional-integral (PI) controller on a DNA reaction system. However, a DNA reaction system is driven by fuel DNA strand consumption, and without a sufficient amount of fuel strands, the molecular PI controller cannot perform normal operations as a concentration regulator. In this study, we developed a design method for a molecular PI control system to regenerate fuel strands by introducing photoresponsive reaction control. To this end, we employed a photoresponsive molecule, azobenzene, to guide the reaction direction forward or backward using light irradiation. We validated our renewable design of the PI controller by numerical simulations based on the reaction kinetics. We also confirmed the proof-of-principle of our renewable design by conducting experiments using a basic DNA circuit.

Xeno nucleic acids (XNAs) having non-ribose scaffolds with unique supramolecular properties.

DNA and RNA have significance as genetic materials, therapeutic potential, and supramolecular properties. Advances in nucleic acid chemistry have enabled large-scale synthesis of DNA and RNA oligonucleotides and oligomers of non-natural nucleic acids, including artificial nucleic acids (xeno nucleic acids; XNAs) with non-ribose scaffolds. In this feature article, we review the chemical structures of XNAs with non-ribose scaffolds, their hybridization abilities, and their unique behaviors with a particular focus on the acyclic XNAs. First, we overview XNAs with non-ribose cyclic scaffolds and then those with acyclic scaffolds by focusing on their hybridization abilities with themselves and with DNA and RNA, and discuss the unexpectedly stable homo-duplex formation of acyclic XNAs. Next, we shed light on our acyclic threoninol nucleic acid (aTNA) and serinol nucleic acid (SNA) and show their helical preferences based on their chirality, then orthogonal control of hybridization and helical amplification of achiral XNAs are demonstrated. Finally, we show non-enzymatic template-directed synthesis of L-aTNA, and the creation of an artificial genetic system with XNAs with non-ribose scaffolds is described as a future prospect.

Color-Changing Fluorescent Barcode Based on Strand Displacement Reaction Enables Simple Multiplexed Labeling.

Fluorescence imaging techniques have contributed to our understanding of various biological phenomenon; however, fluorescence spectral overlap significantly restricts multiplexing capability. Several strategies have been reported to overcome this limitation by utilizing the superior programmability of DNA technologies and nanostructures, but in practice, it remains challenging to achieve broad adoption of these multiplexed detection methods due to the complexities of these DNA designs. Here we report a color-changing fluorescent barcode (CCFB) approach that enables multiple labeling with simple and small nucleic acid structure design based on sequential toehold-mediated strand displacement reaction. The emission color of CCFB can vary in the predetermined sequence so that multiple targets can be detected simultaneously. The CCFB complex is composed of several oligonucleotides, and its color sequence can be easily expanded further. The CCFB approach is easy and time-saving to operate since the irreversible color-changing reaction occurs by simply adding complementary oligonucleotide. We herein developed 27 different CCFB labels, which required only 14 oligonucleotides. We demonstrated that the CCFB system can be used to label multiple targets by attaching CCFB label to polystyrene beads. Moreover, the CCFB can be used to detect intracellular proteins simultaneously when it is attached to antibodies. We expect that this practical platform will be adopted for comprehensive biomolecular imaging in cells.

A Pyrene-Modified Serinol Nucleic Acid Nanostructure Converts the Chirality of Threoninol Nucleic Acids into Circularly Polarized Luminescence Signals.

Herein is reported a circularly polarized luminescent (CPL) probe that can respond to the chirality of nucleic acids. An achiral nanostructure was prepared by the hybridization of symmetric serinol nucleic acid (SNA) containing pyrene-modified residues. When chiral oligomers that were complementary to the SNA were added, they induced helicity into the SNA nanowire. Efficient circular dichroism (CD) signal amplification was observed when pyrene was attached to uracil bases through a rigid alkynyl linker. Both CPL and CD signals were observed; they depended on the chirality of the added acyclic threoninol nucleic acid (aTNA) oligomer. This system can be used to convert the chirality of chiral biomolecules into chiroptical signals.

Perylene-Cy3 FRET System to Analyze Photoactive DNA Structures.

We report a new Förster resonance energy transfer (FRET) system for structural analyses of DNA duplexes using perylene and Cy3 as donor and acceptor, respectively, linked at the termini of a DNA duplex via D-threoninol. Experimentally obtained FRET efficiencies were in good agreement with theoretical values calculated based on canonical B-form DNA. Due to the relatively long Förster radius, this system can be used to analyze large DNA structures, and duplexes containing photo-reactive molecules can be analyzed since perylene can be excited with visible light. The system was used to analyze a DNA duplex containing stilbene, demonstrating that in the region of the stilbene cluster the duplex adopts a ladder-like structure rather than helical one. Upon photodimerization between stilbene residues, FRET efficiencies indicated the reaction does not disturb DNA duplex. This FRET system will be useful for analysis of photoreactions of nucleobases as well as a wide range of nucleic acid structures.

A helical amplification system composed of artificial nucleic acids.

Herein we report an amplification system of helical excess triggered by nucleic acid hybridization for the first time. It is usually impossible to prepare achiral nanostructures composed of nucleic acids because of their intrinsic chirality. We used serinol nucleic acid (SNA) oligomers for the preparation of achiral nanowires because SNA oligomers with symmetrical sequences are achiral. Nanowire formation was confirmed by atomic force microscopy and size exclusion chromatography. When a chiral nucleic acid with a sequence complementary to SNA was added to the nanostructure, helicity was induced and a strong circular dichroism signal was observed. The SNA nanowire could amplify the helicity of chiral nucleic acids through nucleobase stacks. The SNA nanostructures have potential for use as platforms to detect chiral biomolecules under aqueous conditions because SNA can be readily functionalized and is water-soluble.

Design and Hybridization Properties of Acyclic Xeno Nucleic Acid Oligomers.

Xeno nucleic acids (XNAs) are analogues of DNA and RNA that have a non-ribose artificial scaffold. XNAs are possible prebiotic genetic carriers as well as alternative genetic systems in artificial life. In addition, XNA oligomers can be used as biological tools. Acyclic XNAs, which do not have cyclic scaffolds, are attractive due to facile their synthesis and remarkably high nuclease resistance. To maximize the performance of XNAs, a negatively charged backbone is preferable to provide sufficient water solubility; however, acyclic XNAs containing polyanionic backbones suffer from high entropy cost upon duplex formation, because of the high flexibility of the acyclic nature. Herein, we review the relationships between the structure and duplex hybridization properties of various acyclic XNA oligomers with polyanion backbones.