Programmable Computational RNA Droplets Assembled via Kissing-Loop Interaction.

DNA droplets, artificial liquid-like condensates of well-engineered DNA sequences, allow the critical aspects of phase-separated biological condensates to be harnessed programmably, such as molecular sensing and phase-state regulation. In contrast, their RNA-based counterparts remain less explored despite more diverse molecular structures and functions ranging from DNA-like to protein-like features. Here, we design and demonstrate computational RNA droplets capable of two-input AND logic operations. We use a multibranched RNA nanostructure as a building block comprising multiple single-stranded RNAs. Its branches engaged in RNA-specific kissing-loop (KL) interaction enables the self-assembly into a network-like microstructure. Upon two inputs of target miRNAs, the nanostructure is programmed to break up into lower-valency structures that are interconnected in a chain-like manner. We optimize KL sequences adapted from viral sequences by numerically and experimentally studying the base-wise adjustability of the interaction strength. Only upon receiving cognate microRNAs, RNA droplets selectively show a drastic phase-state change from liquid to dispersed states due to dismantling of the network-like microstructure. This demonstration strongly suggests that the multistranded motif design offers a flexible means to bottom-up programming of condensate phase behavior. Unlike submicroscopic RNA-based logic operators, the macroscopic phase change provides a naked-eye-distinguishable readout of molecular sensing. Our computational RNA droplets can be applied to in situ programmable assembly of computational biomolecular devices and artificial cells from transcriptionally derived RNA within biological/artificial cells.

Autonomous assembly and disassembly of gliding molecular robots regulated by a DNA-based molecular controller.

In recent years, there has been a growing interest in engineering dynamic and autonomous systems with robotic functionalities using biomolecules. Specifically, the ability of molecular motors to convert chemical energy to mechanical forces and the programmability of DNA are regarded as promising components for these systems. However, current systems rely on the manual addition of external stimuli, limiting the potential for autonomous molecular systems. Here, we show that DNA-based cascade reactions can act as a molecular controller that drives the autonomous assembly and disassembly of DNA-functionalized microtubules propelled by kinesins. The DNA controller is designed to produce two different DNA strands that program the interaction between the microtubules. The gliding microtubules integrated with the controller autonomously assemble to bundle-like structures and disassemble into discrete filaments without external stimuli, which is observable by fluorescence microscopy. We believe this approach to be a starting point toward more autonomous behavior of motor protein-based multicomponent systems with robotic functionalities.

Phase Angle as a Prognostic Predictor for Physical Function in Fragile Osteoporotic Fracture Patients.

Objectives: This study aimed to determine whether the phase angle is associated with physical function at discharge and discharge destination in patients with osteoporotic fragile fractures. Methods: This retrospective cohort study included patients with fragile osteoporotic fractures who were admitted to a convalescent rehabilitation ward. The phase angle was calculated using a body composition meter and bioelectrical impedance analysis. The primary outcome was the Functional Independence Measure motor (FIM-motor) score at discharge, and the secondary outcome was discharge to home. Multivariate analysis was used to determine the association between phase angle and FIM-motor scores at discharge and discharge to home. Results: The study included 127 patients (108 women, age 81.2 ± 9.7 years). The median phase angle on admission was 4.1° for men and 3.6° for women. The median FIM-motor score at discharge was 83, with 92 (72.4%) patients discharged home and 35 (27.6%) discharged to a destination other than home. Multiple regression analysis adjusted for confounders revealed a significant independent association between the phase angle and FIM-motor score at discharge (ß=0.262, P=0.019). However, no significant association was found between phase angle and discharge destination (odds ratio, 1.350; 95% confidence interval: 0.680-2.670, P=0.391). Conclusions: Phase angle was independently associated with physical function at discharge in patients with fragile osteoporotic fractures. For patients with a reduced phase angle on admission, a multidisciplinary approach, including exercise, nutrition, oral health, and medication, should be implemented to maximize improvement in physical function.

Scalable Synthesis of Planar Macroscopic Lipid-Based Multi-Compartment Structures.

As life evolved, the path from simple single cell organisms to multicellular enabled increasingly complex functionalities. The spatial separation of reactions at the micron scale achieved by cellular structures allowed diverse and scalable implementation in biomolecular systems. Mimicking such spatially separated domains in a scalable approach could open a route to creating synthetic cell-like structured systems. Here, we report a facile and scalable method to create multicellular-like, multi-compartment (MC) structures. Aqueous droplet-based compartments ranging from 50 to 400 µm were stabilized and connected together by hydrophobic layers composed of phospholipids and an emulsifier. Planar centimeter-scale MC structures were formed by droplet deposition on a water interface. Further, the resulting macroscopic shapes were shown to be achieved by spatially controlled deposition. To demonstrate configurability and potential versatility, MC assemblies of both homogeneous and mixed compartment types were shown. Notably, magnetically heterogeneous systems were achieved by the inclusion of magnetic nanoparticles in defined sections. Such structures demonstrated actuated motion with structurally imparted directionality. These novel and functionalized structures exemplify a route toward future applications including compartmentally assembled "multicellular" molecular robots.

Surfactant-Assisted Purification of Hydrophobic DNA Nanostructures.

Purification of functional DNA nanostructures is an essential step in achieving intended functions because misfolded structures and the remaining free DNA strands in a solution can interact and affect their behavior. However, due to hydrophobicity-mediated aggregation, it is difficult to purify DNA nanostructures modified with hydrophobic molecules by conventional methods. Herein, we report the purification of cholesterol-modified DNA nanostructures by using a novel surfactant-assisted gel extraction. The addition of sodium cholate (SC) to the sample solution before structure folding prevented aggregation; this was confirmed by gel electrophoresis. We also found that adding sodium dodecyl sulfate (SDS) to the sample inhibited structural folding. The cholesterol-modified DNA nanostructures prepared with SC were successfully purified by gel extraction, and their ability to bind to the lipid membrane surfaces was maintained. This method will facilitate the purification of DNA nanostructures modified with hydrophobic molecules and expand their applicability in the construction of artificial cell-like systems.

System concentration shift as a regulator of transcription-translation system within liposomes.

Biochemical systems in living cells have their optimum concentration ratio among each constituent element to maintain their functionality. However, in the case of the biochemical system with complex interactions and feedbacks among elements, their activity as a system greatly changes by the concentration shift of the entire system irrespective of the concentration ratio among elements. In this study, by using a transcription-translation (TX-TL) system as the subject, we illustrate the principle of the nonlinear relationship between the system concentration and the activity of the system. Our experiment and simulation showed that shifts of the system concentration of TX-TL by dilution and concentration works as a switch of activity and demonstrated its ability to induce a biochemical system to confer the permeability of small molecules to liposomes. These results contribute to the creation of artificial cells with the switch and provide an insight into the emergence of protocells.

Impact of Rehabilitation Dose on Nutritional Status at Discharge from a Convalescent Rehabilitation Ward in Malnourished Patients with Hip Fracture.

The object of this study is to determine the impact of the rehabilitation dose on the nutritional status at discharge from a convalescent rehabilitation ward in malnourished patients with hip fracture. This retrospective case-control study involved malnourished patients with hip fracture aged 65 years or older who had been admitted to a convalescent rehabilitation ward and whose data were registered in the Japan Rehabilitation Nutrition Database. The primary outcome was nutritional status at discharge. Patients were classified according to whether nutritional status was improved or not at discharge, according to the Mini Nutritional Assessment-Short Form® (MNA-SF) score. The association between improved nutritional status and rehabilitation dose was assessed by a logistic regression analysis. Data were available for 145 patients (27 men, 118 women; mean age 85.1 ± 7.9 years). Daily rehabilitation dose was 109.5 (median 94.6-116.2) min and the MNA-SF score at admission was 5 (median 4-6). Nutritional status was improved in 97 patients and not improved in 48. Logistic regression analysis showed the following factors to be independently associated with nutritional status at discharge: Functional Independence Measure score (OR 1.042, 95% CI 1.016-1.068), energy intake (OR 1.002 CI 1.000-1.004), daily rehabilitation dose (OR 1.023, 95% CI 1.002-1.045), and length of hospital stay (OR 1.026, 95% CI 1.003-1.049). The daily rehabilitation dose in malnourished patients with hip fracture may positively impact nutritional status at discharge.

A large, square-shaped, DNA origami nanopore with sealing function on a giant vesicle membrane.

Intaking molecular information from the external environment is essential for the normal functioning of artificial cells/molecular robots. Herein, we report the design and function of a membrane nanopore using a DNA origami square tube with a cross-section of 100 nm2. When the nanopore is added to a giant vesicle that mimics a cell membrane, the permeation of large external hydrophilic fluorescent molecules is observed. Furthermore, the addition of up to four ssDNA strands enables size-based selective transport of molecules. A controllable artificial nanopore should facilitate the communication between the vesicle components and their environment.

DNA Origami "Quick" Refolding Inside of a Micron-Sized Compartment.

Investigations into the refolding of DNA origami leads to the creation of reconstructable nanostructures and deepens our understanding of the sustainability of life. Here, we report the refolding of the DNA origami structure inside a micron-sized compartment. In our experiments, conventional DNA origami and truss-type DNA origami were annealed and purified to remove the excess staples in a test tube. The DNA origami was then encapsulated inside of a micron-sized compartment of water-in-oil droplets, composed of neutral surfactants. The re-annealing process was then performed to initiate refolding in the compartment. The resulting 100-nm-sized DNA nanostructures were observed using atomic force microscopy (AFM), and the qualities of their structures were evaluated based on their shape. We found that the refolding of the DNA origami structure was favored inside the droplets compared with refolding in bulk solution. The refolded structures were able to fold even under "quick" one-minute annealing conditions. In addition, the smaller droplets (average diameter: 1.2 µm) appeared to be more advantageous for the refolding of the origamis than larger droplets. These results are expected to contribute to understanding the principles of life phenomena based on multimolecular polymer self-assembly in a micron-sized compartment, and for the production and maintenance of artificially designed molecules.

Isothermal amplification of specific DNA molecules inside giant unilamellar vesicles.

An isothermal amplification circuit for specific DNA molecules was implemented in giant unilamellar vesicles. Using this circuit, over 5000-fold amplification of output DNAs was achieved, and the amplification behaviour depended on the concentration of input signal DNAs in a cell-sized compartment. Moreover, initiation of the amplification by photo-stimulation was demonstrated.

Large-Scale Preparation of Giant Vesicles by Squeezing a Lipid-Coated Marshmallow-like Silicone Gel in a Buffer.

Giant vesicles were efficiently produced by squeezing a lipid (l-α-phosphatidylcholine from egg yolk)-coated marshmallow-like flexible macroporous silicone monolith in a buffer. The mean diameter of the obtained vesicles was 2 µm, showing a wide distribution, up to tens of micrometers, which was similar to that of vesicles formed by a natural swelling method. It was possible to prepare vesicle dispersions on a scale from several microliters to several hundred milliliters. A protein synthesis system (PURE system) contained in vesicles prepared using this method functioned effectively. Our absorbing-squeezing method is expected to help in studies that use giant vesicles such as artificial cells and drug delivery systems.

Construction of T-Motif-Based DNA Nanostructures through Enzymatic Reactions.

The most common way to fabricate DNA nanostructures is to mix individually synthesized DNA oligomers in one pot. However, if DNA nanostructures could be produced through enzymatic reactions, they could be applied in various environments, including in vivo. Herein, an enzymatic method developed to construct a DNA nanostructure from a simple motif called a T-motif is reported. A long, repeated structure was replicated from a circular template by rolling circle amplification and then cleaved into T-motif segments by restriction enzymes. These motifs have been successfully assembled into a ladder-like nanostructure without purification or controlled annealing. This approach is widely applicable to constructing a variety of DNA nanostructures through enzymatic reactions.

In vitro transcription-translation using bacterial genome as a template to reconstitute intracellular profile.

In vitro transcription-translation systems (TX-TL) can synthesize most of individual genes encoded in genomes by using strong promoters and translation initiation sequences. This fact raises a possibility that TX-TL using genome as a template can reconstitute the profile of RNA and proteins in living cells. By using cell extracts and genome prepared from different organisms, here we developed a system for in vitro genome transcription-translation (iGeTT) using bacterial genome and cell extracts, and surveyed de novo synthesis of RNA and proteins. Two-dimensional electrophoresis and nano LC-MS/MS showed that proteins were actually expressed by iGeTT. Quantitation of transcription levels of 50 genes for intracellular homeostasis revealed that the levels of RNA synthesis by iGeTT are highly correlated with those in growth phase cells. Furthermore, activity of iGeTT was influenced by transcription derived from genome structure and gene location in genome. These results suggest that intracellular profiles and characters of genome can be emulated by TX-TL using genome as a template.

Revolving Vernier Mechanism Controls Size of Linear Homomultimer.

A new kind of the Vernier mechanism that is able to control the size of linear assembly of DNA origami nanostructures is proposed. The mechanism is realized by mechanical design of DNA origami, which consists of a hollow cylinder and a rotatable shaft in it connected through the same scaffold. This nanostructure stacks with each other by the shape complementarity at its top and bottom surfaces of the cylinder, while the number of stacking is limited by twisting angle of the shaft. Experiments have shown that the size distribution of multimeric assembly of the origami depends on the twisting angle of the shaft; the average lengths of the multimer are decamer, hexamer, and tetramer for 0°, 10°, and 20° twist, respectively. In summary, it is possible to affect the number of polymerization by adjusting the precise shape and movability of a molecular structure.

DNA cytoskeleton for stabilizing artificial cells.

Cell-sized liposomes and droplets coated with lipid layers have been used as platforms for understanding live cells, constructing artificial cells, and implementing functional biomedical tools such as biosensing platforms and drug delivery systems. However, these systems are very fragile, which results from the absence of cytoskeletons in these systems. Here, we construct an artificial cytoskeleton using DNA nanostructures. The designed DNA oligomers form a Y-shaped nanostructure and connect to each other with their complementary sticky ends to form networks. To undercoat lipid membranes with this DNA network, we used cationic lipids that attract negatively charged DNA. By encapsulating the DNA into the droplets, we successfully created a DNA shell underneath the membrane. The DNA shells increased interfacial tension, elastic modulus, and shear modulus of the droplet surface, consequently stabilizing the lipid droplets. Such drastic changes in stability were detected only when the DNA shell was in the gel phase. Furthermore, we demonstrate that liposomes with the DNA gel shell are substantially tolerant against outer osmotic shock. These results clearly show the DNA gel shell is a stabilizer of the lipid membrane akin to the cytoskeleton in live cells.

Stepping operation of a rotary DNA origami device.

We constructed a rotary DNA origami device and tested its stepping operation on a mica substrate by sequential strand displacement with four different sets of signal DNA strands. This work paves the way for building a variety of dynamic rotary DNA nanodevices which respond to multiple signals.

Unzipping and shearing DNA with electrophoresed nanoparticles in hydrogels.

We show electric control of unzipping and shearing dehybridization of a DNA duplex anchored to a hydrogel. Tensile force is applied by electrophoresing (25 V cm-1) gold nanoparticles pulling the DNA duplex. The pulled DNA strand is gradually released from the hydrogel. The unzipping release rate is faster than shearing; for example, 3-fold for a 15 base pair duplex, which helps to design electrically driven DNA devices.

Micrometer-sized molecular robot changes its shape in response to signal molecules.

Rapid progress in nanoscale bioengineering has allowed for the design of biomolecular devices that act as sensors, actuators, and even logic circuits. Realization of micrometer-sized robots assembled from these components is one of the ultimate goals of bioinspired robotics. We constructed an amoeba-like molecular robot that can express continuous shape change in response to specific signal molecules. The robot is composed of a body, an actuator, and an actuator-controlling device (clutch). The body is a vesicle made from a lipid bilayer, and the actuator consists of proteins, kinesin, and microtubules. We made the clutch using designed DNA molecules. It transmits the force generated by the motor to the membrane, in response to a signal molecule composed of another sequence-designed DNA with chemical modifications. When the clutch was engaged, the robot exhibited continuous shape change. After the robot was illuminated with light to trigger the release of the signal molecule, the clutch was disengaged, and consequently, the shape-changing behavior was successfully terminated. In addition, the reverse process-that is, initiation of shape change by input of a signal-was also demonstrated. These results show that the components of the robot were consistently integrated into a functional system. We expect that this study can provide a platform to build increasingly complex and functional molecular systems with controllable motility.

Reversible Gel-Sol Transition of a Photo-Responsive DNA Gel.

Stimuli-responsive DNA gels that can undergo a sol-gel transition in response to photo-irradiation provide a way to engineer functional gel material with fully designed DNA base sequences. We propose an X-shaped DNA motif that turns into a gel by hybridization of self-complementary sticky ends. By embedding a photo-crosslinking artificial base in the sticky-end sequence, repetitive gel-sol transitions are achieved through UV irradiation at different wavelengths. The concentration of the DNA motif necessary for gelation is as low as 40 µm after modification of the geometrical properties of the motif. The physical properties, such as swelling degree and diffusion coefficient, were assessed experimentally.

Trading polymeric microspheres: exchanging DNA molecules via microsphere interaction.

A new class of artificial molecular transport system is constructed by polymeric microspheres. The microspheres are prepared by self-assembly of poly(ethylene glycol)-block-poly(3-dimethyl(methacryloyloxyethyl)ammonium propane sulfonate), PEG-b-PDMAPS, by intermolecular dipole-dipole interaction of sulfobetaine side chains in water. Below the upper critical solution temperature (UCST) of PEG-b-PDMAPS, the microspheres (∼1µm) interact with other microspheres by partial and transit fusion. In order to apply the interaction between microspheres, a 3'-TAMRA-labeled single-stranded DNA oligomer (ssDNA) is encapsulated into a PEG-b-PDMAPS microsphere by thermal treatment. The exchange of ssDNA between microspheres is confirmed by fluorescence resonance energy transfer (FRET) quenching derived from double-stranded formation with complementary 5'-BHQ-2-labeled ssDNA encapsulated in PEG-b-PDMAPS microspheres. The exchange rate of ssDNA is controllable by tuning the composition of the polymer. The contact-dependent transport of molecules can be applied in the areas of microreactors, sensor devices, etc.