Koh-ichiroh Shohda

Self-reproduction of supramolecular giant vesicles combined with the amplification of encapsulated DNA

The construction of a protocell from a materials point of view is important in understanding the origin of life. Both self-reproduction of a compartment and self-replication of an informational substance have been studied extensively, but these processes have typically been carried out independently, rather than linked to one another. Here, we demonstrate the amplification of DNA (encapsulated guest) within a self-reproducible cationic giant vesicle (host). With the addition of a vesicular membrane precursor, we observe the growth and spontaneous division of the giant vesicles, accompanied by distribution of the DNA to the daughter giant vesicles. In particular, amplification of the DNA accelerated the division of the giant vesicles. This means that self-replication of an informational substance has been linked to self-reproduction of a compartment through the interplay between polyanionic DNA and the cationic vesicular membrane. Our self-reproducing giant vesicle system therefore represents a step forward in the construction of an advanced model protocell.

Stabilization of DNA nanostructures by photo-cross-linking

Developing methods for stabilizing DNA nanostructures is a major challenge for next-generation nanofabrication, because stable DNA nanostructures are expected to work as building materials in the bottom-up assembly of functional biomolecules and nano-electronic components. Here we show the availability of cross-linking-type photoreaction with 3-cyanovinylcarbazole nucleosides (CNVKs) on DNA nanostructures. DNA double-crossover AB-staggered (DXAB) tiles including cross-linking molecules, CNVKs, self-assembled into two-dimensional (2D) periodic DNA arrays and were covalently connected by photo-cross-linking. The self-assembled DNA arrays before and after photo-cross-linking have been visualized by high-resolution, tapping mode atomic force microscopy (AFM) in buffer. The improvement of the heat tolerance of photo-cross-linked DNA arrays was confirmed by heating and visualizing the DNA nanostructures. The heat-resistant DNA arrays may expand the potential of DNA as a functional material in biotechnology and nanotechnology.

DNA polymerization on the inner surface of a giant liposome for synthesizing an artificial cell model

We have designed a new artificial cell model consisting of a giant liposome, an enzyme, and DNA conjugated with a cholesterol tag by a poly(ethylene glycol) spacer to characterize the model system. The cholesterol tag of the conjugated molecule was anchored to the inner surface of the giant liposome and the single-stranded DNA unit hybridized with a 100-mer template DNA that was added to the water pool inside the liposome. We found that the DNA unit acted as a primer, DNA polymerization proceeded on the inner surface of the liposome. This reaction was a key step of our cell model. Production of a full-length strand was proved by selective cleavage of the polymerized DNA by a restriction enzyme.

Direct Visualization of DNA Duplex Formation on the Surface of a Giant Liposome

Corrigendum published online September 4, 2003. 10.1002/cbic.200390107.

Heat-resistant DNA tile arrays constructed by template-directed photoligation through 5-carboxyvinyl-2′-deoxyuridine

Template-directed DNA photoligation has been applied to a method to construct heat-resistant two-dimensional (2D) DNA arrays that can work as scaffolds in bottom-up assembly of functional biomolecules and nano-electronic components. DNA double-crossover AB-staggered (DXAB) tiles were covalently connected by enzyme-free template-directed photoligation, which enables a specific ligation reaction in an extremely tight space and under buffer conditions where no enzymes work efficiently. DNA nanostructures created by self-assembly of the DXAB tiles before and after photoligation have been visualized by high-resolution, tapping mode atomic force microscopy in buffer. The improvement of the heat tolerance of 2D DNA arrays was confirmed by heating and visualizing the DNA nanostructures. The heat-resistant DNA arrays may expand the potential of DNA as functional materials in biotechnology and nanotechnology.

Compartment size dependence of performance of polymerase chain reaction inside giant vesicles

The amplification of a 1229 bp template DNA that encoded the green fluorescent protein (GFP) was conducted in cell-sized giant vesicles (GVs, ϕ > 1 µm) using a real-time polymerase chain reaction (PCR) technique. The proportion of PCR-proceeded GV reached up to 16% of all GVs. The dependence of PCR on GV size was elucidated by flow cytometry analysis.

Construction of AND Gate for RTRACS with the Capacity of Extension to NAND Gate

We have succeeded in construction of the AND gate using enzymatic reactions developed for modularized computation elements of the autonomous computing system RTRACS. Experimental results demonstrated that the molecular reaction for the AND gate generated the correct output RNA from input RNAs according to the truth table for the AND gate. The constructed molecular reaction for the AND gate can be extended to the NAND gate by small modifications, because not only a logical 1 but also a logical 0 for inputs and output was associated with the presence of RNA strands.

A DNA based molecular logic gate capable of a variety of logical operations

In this paper we report on a module in the RTRACS (Reverse-transcription and TRanscription-based Autonomous Computing System) molecular computing system, constructed with DNA, RNA and enzymes. The module is a 2-input logic gate that receives input and produces output in the form of RNA molecules. Each of the two input molecules is chosen from a set of two, and the logic gate produces an output molecule for each of the four possible input combinations. Two output RNA molecules can be produced by this module, one for only one combination of inputs, whilst the remaining three combinations lead to the production of the other output. Since the RNA strands can be arbitrarily assigned logical values, this module is capable performing multiple logical operations, including AND, NAND, OR and NOR, given the appropriate mapping of RNA molecules to logical values. We performed numerical simulations of the logic gate reaction scheme, revealing the details of the kinetics of the production of output molecules and the theoretical input–output characteristics. We experimentally demonstrated the proper functioning of the logic gate and the real time production of output molecules. Furthermore, we demonstrate an implementation of the logic gate in diagnosing single nucleotide polymorphism patterns on samples of human genomic DNA. We believe this versatile logic gate has significant advantages as a basic module of RTRACS due to the wide variety of possible logical operations.

A DNA Based Molecular Logic Gate Capable of a Variety of Logical Operations

In this paper we report on a module in the RTRACS molecular computing system, constructed with DNA, RNA and enzymes. The module is a 2-input logic gate that receives input and produces output in the form of RNA molecules. Each of the two input molecules is chosen from a set of two, and the logic gate produces an output molecule for each of the four possible input combinations. Two output RNA molecules can be produced by this module, one for only one combination of inputs, whilst the remaining three combinations lead to the production of the other output. Since the RNA strands can be arbitrarily assigned logical values, this module is capable performing multiple logical operations, including AND, NAND, OR and NOR, given the appropriate mapping of RNA molecules to logical values. We performed numerical simulations of the logic gate reaction scheme, revealing the details of the kinetics of the production of output molecules and the theoretical input-output characteristics. We also experimentally demonstrated the proper functioning of the logic gate, showing the correct formation of intermediate steps, the real time production of output molecules as well as the input-output characteristics of the module. We believe this versatile logic gate has significant advantages as a basic module of RTRACS due to the wide variety of possible logical operations.

A method of gentle hydration to prepare oil-free giant unilamellar vesicles that can confine enzymatic reactions

We report a new and improved method to prepare, by gentle hydration of lipid films, oil-free giant unilamellar vesicles (GUVs), in which enzymatic reactions can be encapsulated. The traditional method of gentle hydration requires very low concentrations of metal ions, whereas enzymatic reactions generally require mono- and divalent metal ions at physiological concentrations. In order to improve the production of oil-free GUVs that can confine enzymatic reactions, we developed a novel method also based on gentle hydration, but in which the precursor lipid film was doped with both 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEGylated lipid) and sugar. Close examination of the size, shape, and lamellarity of vesicles prepared in this manner demonstrated that the process improves the production of oil-free GUVs even at low temperatures and physiological salt concentrations. PEGylated lipid and sugar were found to synergistically improve GUV formation. Finally, we demonstrate the successful enzymatic synthesis of RNA within oil-free GUVs that were prepared on ice.