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Xenonucleic acids (XNAs), which are composed of artificial scaffolds and natural nucleobases, have unique hybridization properties that depend on the scaffold structure. Here, we functionalized the acyclic XNA serinol nucleic acid (SNA) with nonnatural nucleobases. A linear SNA probe functionalized with 5-perylenylethynyl uracil residues showed weak greenish-yellow excimer emission in the absence of target RNA and bright cyan-green monomer emission in the presence of target RNA. Probe hybridization was rapid and enabled the quantitative measurement of RNA with discrimination of single-base mismatches. We also designed a photoresponsive SNA with two 8-pyrenylvinyl adenine (PVA) residues. Irradiation with blue (455 nm) light caused [2 + 2] photocycloaddition between intrastrand PVAs, resulting in the dissociation of the SNA/RNA duplex, whereas irradiation with ultraviolet (340 nm) light induced cycloreversion of the PVA photodimer and SNA/RNA duplex reformation. Using a combination of 8-naphthylvinyl adenine (NVA) and PVA and irradiation with 465 nm, 405 nm, 340 nm, and 300 nm light, orthogonal control of the formation of SNA(NVA-NVA)/RNA and SNA(PVA-NVA)/RNA duplexes was demonstrated. Thus, nucleobase modifications further expand the utility of acyclic XNA in bionanotechnology.

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.

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 acyclicd-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 acyclicl-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.

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 acyclicd-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 acyclicl-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.

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.

A molecular robot is an autonomous micro/nano machine that mounts a molecular controller connecting the sensor and actuator systems. A major control problem of a molecular robot is to maintain the actuator operation at the desired level. This requires a regulator that adjusts the concentration of a specific DNA strand of a system to the desired level to be appropriately implemented on a chemical reaction system made of molecular interactions among DNA strands. However, although there are several research results on DNA concentration regulators, there is a major problem: the regulators normally work only within a limited period because of the finiteness problem of gate and fuel strands. The operating time of the controller is an important control performance index and is a prerequisite for its practical applications. In this study, the design of a renewable DNA concentration regulator simplified to an experimentally feasible level is proposed, inspired by the previously reported azobenzene-based photo-reaction method used for a renewable PID controller. It is shown that the methodology that timely switches the operation between "regulator mode" and "concentration recovery mode" by light irradiation can serve as a solution for the long-time operation of the DNA concentration regulator.

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 realized in-situ annealed 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.

Effective delivery of extraneous molecules into the cytoplasm of the target cells is important for several drug therapies. Previously, we showed effective in vivo transdermal delivery of naked siRNA into skin cells induced by faint electric treatment (ET) iontophoresis, and significant suppression of target mRNA levels (Kigasawa et al., Int. J. Pharm., 2010). This result indicates that electricity promoted the delivery of siRNA into cytoplasm. In the present study, we analyzed the intracellular delivery of naked anti-luciferase siRNA by faint ET, and found that the luciferase activity of cells expressing luciferase was reduced by in vitro ET like in vivo iontophoresis. Cellular uptake of fluorescent-label siRNA was increased by ET, while low temperature exposure, macropinocytosis inhibitor amiloride and caveolae-mediated endocytosis inhibitor filipin significantly prevented siRNA uptake. These results indicate that the cellular uptake mechanism involved endocytosis. In addition, voltage sensitive fluorescent dye DiBAC4 (3) penetration was increased by ET, and the transient receptor potential channel inhibitor SKF96365 reduced siRNA uptake, suggesting that faint ET reduced membrane potentials by changing intracellular ion levels. Moreover, to analyze cytoplasmic delivery, we used in-stem molecular beacon (ISMB), which fluoresces upon binding to target mRNA in the cytoplasm. Surprisingly, cytoplasmic ISMB fluorescence appeared rapidly and homogeneously after ET, indicating that cytoplasmic delivery is markedly enhanced by ET. In conclusion, we demonstrated for the first time that faint ET can enhance cellular uptake and cytoplasmic delivery of extraneous molecules.