Hirohisa Ohno

Synthetic RNA-protein complex shaped like an equilateral triangle

Synthetic nanostructures consisting of biomacromolecules such as nucleic acids have been constructed using bottom-up approaches. In particular, Watson-Crick base pairing has been used to construct a variety of two- and three-dimensional DNA nanostructures. Here, we show that RNA and the ribosomal protein L7Ae can form a nanostructure shaped like an equilateral triangle that consists of three proteins bound to an RNA scaffold. The construction of the complex relies on the proteins binding to kink-turn (K-turn) motifs in the RNA, which allows the RNA to bend by ∼ 60° at three positions to form a triangle. Functional RNA-protein complexes constructed with this approach could have applications in nanomedicine and synthetic biology.

Cell-surface receptor control that depends on the size of a synthetic equilateral-triangular RNA-protein complex

A human cell surface displays many complex-structured receptors for receiving extracellular signals to regulate cellular functions. The use of precisely regulated signal-controls of the receptors could have possibilities beyond the current synthetic biology research that begins with the transfection of exogenous molecules to rewire intracellular circuits. However, by using a current ligand-receptor technique, the configuration of the artificially assembled cell surface molecules has been undefined because the assemblage is an unsystematic molecular clustering. Thus, the system bears improvements for precisely regulating receptor functions. We report here a new tool that refines stereochemically-controlled positioning of an assembled surface receptor. The tool performs rationally as an ON/OFF switch and is finely tunable so that a 3 to 6 nm size difference of the device precisely distinguishes the efficiency of apoptosis induced via cell-surface receptor binding. We discuss the potential use of the device in next-generation synthetic biology and in cell surface studies.

Designed Regular Tetragon-Shaped RNA-Protein Complexes with Ribosomal Protein L1 for Bionanotechnology and Synthetic Biology.

RNA nanotechnology has been established by employing the molecular architecture of RNA structural motifs. Here, we report two designed RNA-protein complexes (RNPs) composed of ribosomal protein L1 (RPL1) and its RNA-binding motif that are square-shaped nano-objects. The formation and the shape of the objects were confirmed by gel electrophoresis analysis and atomic force microscopy, respectively. Any protein can be attached to the RNA via a fusion protein with RPL1, indicating that it can be used as a scaffold for loading a variety of functional proteins or for building higher-order structures. In summary, the RNP object will serve as a useful tool in the fields of bionanotechnology and synthetic biology. Moreover, the RNP interaction enhances the RNA stability against nucleases, rendering these complexes stable in cells.

A Purification Method for a Molecular Complex in Which a Scaffold Molecule Is Fully Loaded with Heterogeneous Molecules

An affinity resin-based pull-down method is convenient for the purification of biochemical materials. However, its use is difficult for the isolation of a molecular complex fully loaded with multiple components from a reaction mixture containing the starting materials and intermediate products. To overcome this problem, we have developed a new purification procedure that depends on sequential elimination of the residues. In practice, two affinity resins were used for purifying a triangular-shaped RNP (RNA-protein complex) consisting of three ribosomal proteins (L7Ae) bound to an RNA scaffold. First, a resin with immobilized L7Ae protein captured the incomplete RNP complexes and the free RNA scaffold. Next, another resin with an immobilized chemically modified RNA of a derivative of Box C/D motif, the binding partner of L7Ae, was used to capture free protein. The complete triangular RNP was successfully purified from the mixture by these two steps. Obviously, the purified triangular RNP displaying three protein-binding peptides exhibited an improved performance when compared with the unrefined product. Conceptually, this purification procedure should be applicable for the purification of a variety of complexes consisting of multiple components other than RNP.

Protein-driven RNA nanostructured devices that function in vitro and control mammalian cell fate

Nucleic acid nanotechnology has great potential for future therapeutic applications. However, the construction of nanostructured devices that control cell fate by detecting and amplifying protein signals has remained a challenge. Here we design and build protein-driven RNA-nanostructured devices that actuate in vitro by RNA-binding-protein-inducible conformational change and regulate mammalian cell fate by RNA–protein interaction-mediated protein assembly. The conformation and function of the RNA nanostructures are dynamically controlled by RNA-binding protein signals. The protein-responsive RNA nanodevices are constructed inside cells using RNA-only delivery, which may provide a safe tool for building functional RNA–protein nanostructures. Moreover, the designed RNA scaffolds that control the assembly and oligomerization of apoptosis-regulatory proteins on a nanometre scale selectively kill target cells via specific RNA–protein interactions. These findings suggest that synthetic RNA nanodevices could function as molecular robots that detect signals and localize target proteins, induce RNA conformational changes, and programme mammalian cellular behaviour.Nucleic acid nanotechnology has great potential for future therapeutic applications. Here the authors build protein-driven RNA nanostructures that can function within mammalian cells and regulate the cell fate.

Design, Assembly, and Evaluation of RNA–Protein Nanostructures

The use of RNA-protein interaction motifs (RNP motifs) to design and build nanoscale objects has the potential to expand the field of RNA nanotechnology. In principle, RNP motifs can be integrated easily into RNA nano objects, providing an alternative technique to increase the functional and structural complexities of the RNA. Investigating the design principles of RNP nanostructures will enable the construction of highly sophisticated biomacromolecular complexes such as ribosomes from scratch. As an initial step towards this goal, we designed and constructed triangular-like nanostructures by employing box C/D kink-turn (K-turn)-L7Ae RNP motifs. We showed that the K-turn RNA and the ribosomal protein L7Ae could form a nanostructure shaped like an equilateral triangle that consists of the three proteins attached to the tips of the RNA scaffold. The construction of the complex depends on L7Ae binding to the K-turn motifs in the RNA. The RNP motif allows the RNA to bend by approximately 60° at three positions to form a nanoscale triangle. Functional RNP triangles with desired protein modules at the three tips can be constructed in a modular manner. Here, we describe how to design, construct, and evaluate the RNP nanostructures.