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.

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.

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.

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.

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.

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.

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.

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 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.

Microheater-integrated zinc oxide nanowire microfluidic device for hybridization-based detection of target single-stranded DNA.

Detection of cell-free DNA (cfDNA) has an impact on DNA analysis in liquid biopsies. However, current strategies to detect cfDNA have limitations that should be overcome, such as having low sensitivity and requiring much time and a specialized instrument. Thus, non-invasive and rapid detection tools are needed for disease prevention and early-stage treatment. Here we developed a device having a microheater integrated with zinc oxide nanowires (microheater-ZnO-NWs) to detect target single-stranded DNAs (ssDNAs) based on DNA probe hybridization. We confirmed experimentally that our device realizedin-situannealed DNA probes by which we subsequently detected target ssDNAs. We envision that this device can be utilized for fundamental studies related to nanobiodevice-based DNA detection.

Efficient Light-Harvesting Antennae Resulting from the Dense Organization of Dyes into DNA Junctions through d-Threoninol.

Herein we report the construction of efficient light-harvesting antennae by hybridization of DNA oligonucleotides containing high densities of fluorophores into DNA junctions through d-threoninol. Six pyrene donors could be incorporated into each arm without self-quenching. A perylene acceptor was located at the center of the junction. Antenna effects of a duplex and three- to eight-way junctions were systematically compared. Six- and eight-way junctions had the highest antenna effects, and their effective absorption coefficients were 8.5 times higher than that of perylene. Interestingly, even-numbered junctions had higher efficiencies than odd-numbered junctions. Nondenaturing gel analyses and fluorescence lifetime measurements demonstrated that the strong odd-even effects were derived from differences in the stability of junctions. The results presented will guide the design of efficient artificial photosynthetic systems.

Selective binding of nucleosides to gapped DNA duplex revealed by orientation and distance dependence of FRET.

Herein we used orientation and distance dependence of Förster resonance energy transfer (FRET) to analyze the binding of nucleosides to a gapped DNA duplex. Binding isotherms and information on the structures of the complexes were obtained by monitoring FRET between pyrene and perylene, which were introduced into the DNA through d-threoninol. FRET efficiency significantly changed upon formation of a duplex with a 1-nucleotide gap and a nucleoside. The FRET plot indicated that the complex has a double helical structure similar to a nicked duplex. Cooperative binding of two nucleosides to a duplex with a 2-nucleotide gap was also revealed using FRET. Various drug-nucleic acids interactions could be investigated using this sensitive and facile method.

Orientation-dependent FRET system reveals differences in structures and flexibilities of nicked and gapped DNA duplexes.

Differences in structures and flexibilities of DNA duplexes play important roles on recognition by DNA-binding proteins. We herein describe a novel method for structural analyses of DNA duplexes by using orientation dependence of Förster resonance energy transfer (FRET). We first analyzed canonical B-form duplex and correct structural parameters were obtained. The experimental FRET efficiencies were in excellent agreement with values theoretically calculated by using determined parameters. We then investigated DNA duplexes with nick and gaps, which are key intermediates in DNA repair systems. Effects of gap size on structures and flexibilities were successfully revealed. Since our method is facile and sensitive, it could be widely used to analyze DNA structures containing damages and non-natural molecules.

Bifacial Nucleobases for Hexaplex Formation in Aqueous Solution.

Although DNA can form triplex and quadruplex structures through hydrogen bonds, design and preparation of structures with more than five strands is difficult even when artificial nucleic acids are used. Herein we report a hexaplex formed by oligomers of artificial nucleic acids bearing bifacial molecules on d-threoninol. Aminopyrimidine and cyanuric acid derivatives were selected as bases because they have complementary hydrogen bonding patterns. The complex formed by aminopyrimidine and cyanuric acid decamers melted with large hysteresis. Hexaplex formation was indicated by gel electrophoresis, size exclusion chromatography and atomic force microscopy imaging, and proven directly through native mass spectrometry. CD measurements and molecular dynamics simulations indicated that the hexaplex adopts a helical structure. The hexaplex formation was highly dependent on pH and the presence of divalent cations. The hexaplex was stable in aqueous solution, and its unique structure and properties may lead to novel nanostructures, molecular assemblies, metal sensors, and ion channels.

Chaperone-Polymer-Assisted, Photodriven DNA Strand Displacement.

Photodriven DNA strand displacement by using a 2',6'-dimethylazobenzene-tethered strand and poly(l-lysine)-graft-dextran (PLL-g-Dex) as a chaperone is reported. Rapid strand displacement was reversibly induced by UV and visible-light irradiation without any toehold portion. To further improve the method, the concentration of PLL-g-Dex and the number of equivalents of the photoresponsive strand were optimised. Optimally, 64 % strand displacement was reversibly induced by alternating UV and visible-light irradiation.

Visible-Light-Triggered Cross-Linking of DNA Duplexes by Reversible [2+2] Photocycloaddition of Styrylpyrene.

Reversible photo-cross-linking of a DNA duplex through the [2+2] photocycloaddition of styrylpyrene is reported. Styrylpyrene moieties on d-threoninol linkers were introduced into complementary positions on DNA strands. Irradiation of the styrylpyrene pair in the duplex with visible light at λ=455 nm induced a [2+2] photocycloaddition between styrylpyrenes that cross-linked the two strands of the duplex. Two diastereomers were formed after [2+2] photocycloaddition as a result of rotation of the styrylpyrene residues. Also, the cycloreversion reaction was induced by UV light at λ=340 nm, which reversibly yielded the uncross-linked strands.

Strand-invading linear probe combined with unmodified PNA.

Efficient strand invasion by a linear probe to fluorescently label double-stranded DNA has been implemented by employing a probe and unmodified PNA. As a fluorophore, we utilized ethynylperylene. Multiple ethynylperylene residues were incorporated into the DNA probe via a d-threoninol scaffold. The ethynylperylene did not significantly disrupt hybridization with complementary DNA. The linear probe self-quenched in the absence of target DNA and did not hybridize with PNA. A gel-shift assay revealed that linear probe and PNA combination invaded the central region of double-stranded DNA upon heat-shock treatment to form a double duplex. To further suppress the background emission and increase the stability of the probe/DNA duplex, a probe containing anthraquinones as well as ethynylperylene was synthesized. This probe and PNA invader pair detected an internal sequence in a double-stranded DNA with high sensitivity when heat shock treatment was used. The probe and PNA pair was able to invade at the terminus of a long double-stranded DNA at 40°C at 100mM NaCl concentration.

A stem-less probe using spontaneous pairing between Cy3 and quencher for RNA detection.

We herein report a stem-less probe for the detection of RNA that depends on pairing between Cy3 and nitro methyl red. In our design, two Cy3 residues and two nitro methyl red residues were introduced into an oligonucleotide. In the absence of the target, these dyes formed a complex, and emission of Cy3 was efficiently quenched. Hybridization with the target RNA disrupted this interaction and resulted in Cy3 emission. Under optimized conditions, the signal to background ratio was as high as 180. We demonstrated specific detection of target RNA in cells using a wash-free FISH protocol.

Ultrasensitive Molecular Beacon Designed with Totally Serinol Nucleic Acid (SNA) for Monitoring mRNA in Cells.

An artificial nucleic acid based on acyclic serinol building blocks and termed "serinol nucleic acid" (SNA) was used to construct a fluorescent probe for RNA visualization in cells. The molecular beacon (MB) composed of only SNA with a fluorophore at one terminus and a quencher at the other was resistant to enzymatic digestion, due to its unnatural acyclic scaffold. The SNA-MB could detect its complementary RNA with extremely high sensitivity; the signal-to-background (S/B) ratio was as high as 930 when perylene and anthraquinone were used as the fluorophore and quencher pair. A high S/B ratio was also achieved with SNA-MB tethering the conventional Cy3 fluorophore, and this probe enabled selective visualization of target mRNA in fixed cells. Thus, SNA-MB has potential for use as a biological tool capable of visualizing RNA in living cells.

Hetero-Selective DNA-Like Duplex Stabilized by Donor-Acceptor Interactions.

We report on the characterization of a novel hetero-selective DNA-like duplex of pyrene and anthraquinone pseudo base pairs. The pyrene/anthraquinone pairs showed excellent selectivity in hetero-recognition and even trimers were found to form a hetero-duplex. Pyrene and anthraquinone moieties were tethered on acyclic D-threoninol linkers and linked to adjacent residues by using standard phosphoramidite chemistry. When pyrene and anthraquinone were incorporated at pairing positions in complementary strands of natural DNA oligonucleotides, the duplex was stabilized significantly. Moreover, a pyrene hexamer and an anthraquinone hexamer formed a stable artificial hetero-duplex without the assistance of natural base pairs. The pyrene/anthraquinone pair was so stable that even trimers formed a hetero-duplex under conditions in which natural DNA strands of three residues do not.