Hirohide Saito

An in vitro evolved precursor tRNA with aminoacylation activity

A set of catalysts for aminoacyl‐tRNA synthesis is an essential component for translation. The RNA world hypothesis postulates that RNA catalysts could have played this role. Here we show an in vitro evolved precursor tRNA consisting of two domains, a catalytic 5′‐leader sequence and an aminoacyl‐acceptor tRNA. The 5′‐leader sequence domain selectively self‐charges phenylalanine on the 3′‐terminus of the tRNA domain. This cis‐acting ribozyme is susceptible to RNase P RNA, generating the corresponding 5′‐leader segment and the mature tRNA. Moreover, the 5′‐leader segment is able to aminoacylate the mature tRNA in trans. Mutational studies have revealed that C74 and C75 at the tRNA aminoacyl‐acceptor end form base pairs with G71 and G70 of the trans‐acting ribozyme. Such Watson–Crick base pairing with tRNA has been observed in RNase P RNA and 23S rRNA, suggesting that all three ribozymes use a similar mechanism for the recognition of the aminoacyl‐acceptor end. Our demonstrations indicate that catalytic precursor tRNAs could have provided the foundations for the genetic coding system in the proto‐translation system.

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.

Synthetic translational regulation by an L7Ae–kink-turn RNP switch

The regulation of cell signaling pathways and the reconstruction of genetic circuits are important aspects of bioengineering research. Both of these goals require molecular devices to transmit information from an input biomacromolecule to the desired outputs. Here, we show that an RNA-protein (RNP)-containing L7Ae-kink-turn interaction can be used to construct translational regulators under control of an input protein that regulates the expression of desired output proteins. We built a system in which L7Ae, an archaeal ribosomal protein, regulates the translation of a designed mRNA in vitro and in human cells. The translational regulator composed of the RNP might provide new therapeutic strategies based on the detection, repair or rewiring of intrinsic cellular defects, and it may also serve as an invaluable tool for the dissection of the behavior of complex, higher-order circuits in the cell.

A Versatile tRNA Aminoacylation Catalyst Based on RNA

Aminoacyl-tRNA synthetase (ARS) ribozymes have potential to develop a novel genetic coding system. Although we have previously isolated such a ribozyme that recognizes aromatic amino acids, it could not be used as a versatile catalyst due to its limited ability of aminoacylation to a particular tRNA used for the selection. To overcome this limitation, we used a combination of evolutionary and engineering approaches to generate an optimized ribozyme. The ribozyme, consisting of 45 nucleotides, displays a broad spectrum of activity toward various tRNAs. Most significantly, this ribozyme is able to exhibit multiple turnover activity and charge parasubstituted Phe analogs onto an engineered suppressor tRNA (tRNA(Asn)(CCCG)). Thus, it provides a useful and flexible tool for the custom synthesis of mischarged tRNAs with natural and nonnatural amino acids.

Efficient Detection and Purification of Cell Populations Using Synthetic MicroRNA Switches

Isolation of specific cell types, including pluripotent stem cell (PSC)-derived populations, is frequently accomplished using cell surface antigens expressed by the cells of interest. However, specific antigens for many cell types have not been identified, making their isolation difficult. Here, we describe an efficient method for purifying cells based on endogenous miRNA activity. We designed synthetic mRNAs encoding a fluorescent protein tagged with sequences targeted by miRNAs expressed by the cells of interest. These miRNA switches control their translation levels by sensing miRNA activities. Several miRNA switches (miR-1-, miR-208a-, and miR-499a-5p-switches) efficiently purified cardiomyocytes differentiated from human PSCs, and switches encoding the apoptosis inducer Bim enriched for cardiomyocytes without cell sorting. This approach is generally applicable, as miR-126-, miR-122-5p-, and miR-375-switches purified endothelial cells, hepatocytes, and insulin-producing cells differentiated from hPSCs, respectively. Thus, miRNA switches can purify cell populations for which other isolation strategies are unavailable.

Time-Resolved Tracking of a Minimum Gene Expression System Reconstituted in Giant Liposomes

Individual expression: We describe a method that allows the observation of real‐time gene expression in a large number of individual giant liposomes encapsulating identical genetic material. We followed the gene expression profiles from DNA and mRNA templates coding for different proteins. Although the average profiles of individual liposomes were similar to those measured in bulk solution, strong variability between individual liposomes was observed at both transcription and translation.

Molecular Robotics: A New Paradigm for Artifacts

The rapid progress of molecular nanotechnology has opened the door to molecular robotics, which uses molecules as robot components. In order to promote this new paradigm, the Molecular Robotics Research Group was established in the Systems and Information Division of the Society of Instrument and Control Engineers (SICE) in 2010. The group consists of researchers from various fields including chemistry, biophysics, DNA nanotechnology, systems science and robotics, challenging this emerging new field. Last year, the group proposed a research project focusing on molecular robotics, and it was recently awarded a Grant-in-Aid for Scientific Research on Innovative Areas (FY2012-16), one of the large-scale research projects in Japan, by MEXT (Ministry of Education, Culture, Sports, Science and Technology, JAPAN). Here, we wish to clarify the fundamental concept and research direction of molecular robotics. For this purpose, we present a comprehensive view of molecular robotics based on the discussions held in the Molecular Robotics Research Group.

Synthetic human cell fate regulation by protein-driven RNA switches

Understanding how to control cell fate is crucial in biology, medical science and engineering. In this study, we introduce a method that uses an intracellular protein as a trigger for regulating human cell fate. The ON/OFF translational switches, composed of an intracellular protein L7Ae and its binding RNA motif, regulate the expression of a desired target protein and control two distinct apoptosis pathways in target human cells. Combined use of the switches demonstrates that a specific protein can simultaneously repress and activate the translation of two different mRNAs: one protein achieves both up- and downregulation of two different proteins/pathways. A genome-encoded protein fused to L7Ae controlled apoptosis in both directions (death or survival) depending on its cellular expression. The method has potential for curing cellular defects or improving the intracellular production of useful molecules by bypassing or rewiring intrinsic signal networks.

Synthetic biology with RNA motifs

Structural motifs in naturally occurring RNAs and RNPs can be employed as new molecular parts for synthetic biology to facilitate the development of novel devices and systems that modulate cellular functions. In this review, we focus on the following: (i) experimental evolution techniques of RNA molecules in vitro and (ii) their applications for regulating gene expression systems in vivo. For experimental evolution, new artificial RNA aptamers and RNA enzymes (ribozymes) have been selected in vitro. These functional RNA molecules are likely to be applicable in the reprogramming of existing gene regulatory systems. Furthermore, they may be used for designing hypothetical RNA-based living systems in the so-called RNA world. For the regulation of gene expressions in living cells, the development of new riboswitches allows us to modulate the target gene expression in a tailor-made manner. Moreover, recently RNA-based synthetic genetic circuits have been reported by employing functional RNA molecules, expanding the repertory of synthetic biology with RNA motifs.

Feedback control of protein expression in mammalian cells by tunable synthetic translational inhibition.

Feedback regulation plays a crucial role in dynamic gene expression in nature, but synthetic translational feedback systems have yet to be demonstrated. Here we use an RNA/protein interaction-based synthetic translational switch to create a feedback system that tightly controls the expression of proteins of interest in mammalian cells. Feedback is mediated by modified ribosomal L7Ae proteins, which bind a set of RNA motifs with a range of affinities. We designed these motifs into L7Ae-encoding mRNA. Newly translated L7Ae binds its own mRNA, inhibiting further translation. This inhibition tightly feedback-regulates the concentration of L7Ae and any fusion partner of interest. A mathematical model predicts system behavior as a function of RNA/protein affinity. We further demonstrate that the L7Ae protein can simultaneously and tunably regulate the expression of multiple proteins of interest by binding RNA control motifs built into each mRNA, allowing control over the coordinated expression of protein networks.