The aim of our project is the construction of an autonomous micrometer-sized molecular robot. We designed and constructed a micrometer-sized molecular robot, “DNA ciliate”, named after water microorganisms, ciliate. The DNA ciliate has three independent functional modes (see the left figure): free moving mode, track walking mode, and light-irradiated gathering mode.

The DNA ciliate consists of a micrometer-sized body and cilia. The micrometer-sized body is made of a microsphere such as a polystyrene microbead, a glass microbead, etc. The cilia are made of deoxyribozymes (single-stranded DNAs with cleaving activity). In the free moving mode, the DNA ciliate move around in a broad space based on the Brownian motion. In addition, the DNA ciliate can walk along a single-stranded DNA track using the deoxyribozyme activity. The DNA ciliate can be gathered up at a UV-irradiated area. The construction of the DNA ciliate is shown in detail below.

The DNA ciliate consists of a micrometer-sized body and cilia. The body of the DNA ciliate is made of a polystyrene microbead with a diameter of about 1 μm. The cilia made of deoxyribozymes are attached on the surface of the body with the amide bond (a chemical covalent bond).

The DNA ciliate is an autonomously moving molecular robot. The first moving mode is the free moving based on the Brownian motion in an aqueous media. By the free moving mode, the DNA ciliate can move around and explore in a broad range of space. This motion is similar to a random motion of microorganisms when the microorganisms search food.

The movement by Brownian motion is described by the right equation (1). The left side of the equation is the mean square displacement of a DNA ciliate. R is the displacement from a default position during the time Δt. The diffusion constant D is described by the right equation (2), where T is a temperature, N_A is the Avogadro number, η is a viscosity coefficient, and a is the radius of the DNA ciliate body. The diffusion constant is inversely proportional to the radius of the body. Thus, a DNA ciliate with a smaller body can move more broadly.

In the track walking mode, the DNA ciliate walks directionally along a track. The track is constructed from arrayed single-stranded DNA molecules that are substrates for the deoxyribozymes attached on the DNA ciliate body. DNA tracks with arbitrary shapes can be constructed using DNA nanotechnology, microfluidic technology, etc.

The mechanism of track walking

The cleaving activity of the deoxyribozymes on the DNA ciliate body is used when the DNA ciliates work at the track walking mode. The reaction scheme of the track walking is presented in the right figure. First, a deoxyribozyme hybridizes with a substrate DNA composing a track. The deoxyribozyme has an RNA cleaving activity and cleaves a ribose site (yellow site in the right figure) included in the substrate DNA. After the cleavage, the cleaved and shorten substrate and the deoxyribozyme dissociate because the thermodynamically stability between the deoxyribozyme and the shorten substrate DNAs decreases. The dissociated deoxyribozyme can hybridize with another uncleaved substrate DNA near the cleaved substrate DNA. The deoxyribozyme more easily hybridizes with an uncleaved substrate than a cleaved one. Thus, the DNA ciliate can walk along the DNA track by repeating this reaction.

Construction of DNA tracks

Although nanometer-sized DNA machines such as a DNA spider walks along a nanometer-sized DNA track constructed from DNA origami and DNA nanostructure, the DNA ciliate walks along a micrometer-sized DNA track (shown in the right figure) because the DNA ciliate has a micrometer-sized body. The micrometer-sized DNA tracks can be constructed using DNA nanotechnology, microfluidic technology, etc. Here, the micrometer-sized DNA tracks were constructed using a microchannel. Substrate DNAs were immobilized on a glass plate as a self-assembled monolayer through a silane coupling reaction.

In the light-irradiated gathering mode, DNA ciliates gather at a specific UV-irradiated UV area; i.e., the DNA ciliate can be controlled by UV irradiation. This mode is achieved by a UV-switching DNA device with UV-responsive bases, azobenzenes. Before UV-irradiattion, this DNA device does not hybridize with deoxyribozyme of the DNA ciliate. After UV irradiation, azobenzenes are isomerized and the DNA device can hybridize with the deoxyribozyme of DNA ciliate. By this reaction, the hybridization of DNA ciliates with arrayed DNAs on a glass plate is controlled.

UV-switching DNA device and the mechanism of light-irradiated gathering

The UV-switching DNA device is composed of a UV-switching DNA and a blocking DNA. The UV-switching DNA includes a complementary sequence of the deoxyribozyme on the DNA ciliate. However, the deoxyribozyme cannot hybridize with the UV-switching DNA before UV irradiation because the UV-switching DNA has a stem-loop structure and the blocking DNA at the complementary sequence of the deoxyribozyme (the “closed” state in the right figure). After UV irradiation, the stem-loop structure of the UV-switching DNA open, caused by the UV-responsive structural transition of azobenzenes in the UV-switching DNA. When the UV-switching DNA is open state, the deoxyribozyme can partly hybridizes with the UV-switching DNA and finally wholly hybridizes with the UV-switching DNA as a result of the branch migration of them.