Deep generative design of RNA family sequences.

RNA engineering has immense potential to drive innovation in biotechnology and medicine. Despite its importance, a versatile platform for the automated design of functional RNA is still lacking. Here, we propose RNA family sequence generator (RfamGen), a deep generative model that designs RNA family sequences in a data-efficient manner by explicitly incorporating alignment and consensus secondary structure information. RfamGen can generate novel and functional RNA family sequences by sampling points from a semantically rich and continuous representation. We have experimentally demonstrated the versatility of RfamGen using diverse RNA families. Furthermore, we confirmed the high success rate of RfamGen in designing functional ribozymes through a quantitative massively parallel assay. Notably, RfamGen successfully generates artificial sequences with higher activity than natural sequences. Overall, RfamGen significantly improves our ability to design functional RNA and opens up new potential for generative RNA engineering in synthetic biology.

Synthetic circular RNA switches and circuits that control protein expression in mammalian cells.

Synthetic messenger RNA (mRNA) has been focused on as an emerging application for mRNA-based therapies and vaccinations. Recently, synthetic circular RNAs (circRNAs) have shown promise as a new class of synthetic mRNA that enables superior stability and persistent gene expression in cells. However, translational control of circRNA remained challenging. Here, we develop 'circRNA switches' capable of controlling protein expression from circRNA by sensing intracellular RNA or proteins. We designed microRNA (miRNA) and protein-responsive circRNA switches by inserting miRNA-binding or protein-binding sequences into untranslated regions (UTRs), or Coxsackievirus B3 Internal Ribosome Entry Site (CVB3 IRES), respectively. Engineered circRNAs efficiently expressed reporter proteins without inducing severe cell cytotoxicity and immunogenicity, and responded to target miRNAs or proteins, controlling translation levels from circRNA in a cell type-specific manner. Moreover, we constructed circRNA-based gene circuits that selectively activated translation by detecting endogenous miRNA, by connecting miRNA and protein-responsive circRNAs. The designed circRNA circuits performed better than the linear mRNA-based circuits in terms of persistent expression levels. Synthetic circRNA devices provide new insights into RNA engineering and have a potential for RNA synthetic biology and therapies.

Versatile strategy using vaccinia virus-capping enzyme to synthesize functional 5' cap-modified mRNAs.

The potential of synthetic mRNA as a genetic carrier has increased its application in scientific fields. Because the 5' cap regulates the stability and translational activity of mRNAs, there are concerted efforts to search for and synthesize chemically-modified 5' caps that improve the functionality of mRNA. Here, we report an easy and efficient method to synthesize functional mRNAs by modifying multiple 5' cap analogs using a vaccinia virus-capping enzyme. We show that this enzyme can introduce a variety of GTP analogs to the 5' end of RNA to generate 5' cap-modified mRNAs that exhibit different translation levels. Notably, some of these modified mRNAs improve translation efficiency and can be conjugated to chemical structures, further increasing their functionality. Our versatile method to generate 5' cap-modified mRNAs will provide useful tools for RNA therapeutics and biological research.

Sensing intracellular signatures with synthetic mRNAs.

The bottom-up assembly of biological components in synthetic biology has contributed to a better understanding of natural phenomena and the development of new technologies for practical applications. Over the past few decades, basic RNA research has unveiled the regulatory roles of RNAs underlying gene regulatory networks; while advances in RNA biology, in turn, have highlighted the potential of a wide variety of RNA elements as building blocks to construct artificial systems. In particular, synthetic mRNA-based translational regulators, which respond to signals in cells and regulate the production of encoded output proteins, are gaining attention with the recent rise of mRNA therapeutics. In this Review, we discuss recent progress in RNA synthetic biology, mainly focusing on emerging technologies for sensing intracellular protein and RNA molecules and controlling translation.

Xeno-free induced pluripotent stem cell-derived neural progenitor cells for in vivo applications.

BACKGROUND: Currently, there is no regenerative therapy for patients with neurological and neurodegenerative disorders. Cell-therapies have emerged as a potential treatment for numerous brain diseases. Despite recent advances in stem cell technology, major concerns have been raised regarding the feasibility and safety of cell therapies for clinical applications. METHODS: We generated good manufacturing practice (GMP)-compatible neural progenitor cells (NPCs) from transgene- and xeno-free induced pluripotent stem cells (iPSCs) that can be smoothly adapted for clinical applications. NPCs were characterized in vitro for their differentiation potential and in vivo after transplantation into wild type as well as genetically immunosuppressed mice. RESULTS: Generated NPCs had a stable gene-expression over at least 15 passages and could be scaled for up to 1018 cells per initially seeded 106 cells. After withdrawal of growth factors in vitro, cells adapted a neural fate and mainly differentiated into active neurons. To ensure a pure NPC population for in vivo applications, we reduced the risk of iPSC contamination by applying micro RNA-switch technology as a safety checkpoint. Using lentiviral transduction with a fluorescent and bioluminescent dual-reporter construct, combined with non-invasive in vivo bioluminescent imaging, we longitudinally tracked the grafted cells in healthy wild-type and genetically immunosuppressed mice as well as in a mouse model of ischemic stroke. Long term in-depth characterization revealed that transplanted NPCs have the capability to survive and spontaneously differentiate into functional and mature neurons throughout a time course of a month, while no residual pluripotent cells were detectable. CONCLUSION: We describe the generation of transgene- and xeno-free NPCs. This simple differentiation protocol combined with the ability of in vivo cell tracking presents a valuable tool to develop safe and effective cell therapies for various brain injuries.

N 1-Methylpseudouridine substitution enhances the performance of synthetic mRNA switches in cells.

Synthetic messenger RNA (mRNA) tools often use pseudouridine and 5-methyl cytidine as substitutions for uridine and cytidine to avoid the immune response and cytotoxicity induced by introducing mRNA into cells. However, the influence of base modifications on the functionality of the RNA tools is poorly understood. Here we show that synthetic mRNA switches containing N1-methylpseudouridine (m1Ψ) as a substitution of uridine substantially out-performed all other modified bases studied, exhibiting enhanced microRNA and protein sensitivity, better cell-type separation ability, and comparably low immune stimulation. We found that the observed phenomena stem from the high protein expression from m1Ψ containing mRNA and efficient translational repression in the presence of target microRNAs or proteins. In addition, synthetic gene circuits with m1Ψ significantly improve performance in cells. These findings indicate that synthetic mRNAs with m1Ψ modification have enormous potentials in the research and application of biofunctional RNA tools.

Nanoparticle-based local translation reveals mRNA as a translation-coupled scaffold with anchoring function.

The spatial regulation of messenger RNA (mRNA) translation is central to cellular functions and relies on numerous complex processes. Biomimetic approaches could bypass these endogenous complex processes, improve our comprehension of the regulation, and allow for controlling local translation regulations and functions. However, the causality between local translation and nascent protein function remains elusive. Here, we developed a nanoparticle (NP)-based strategy to magnetically control mRNA spatial patterns in mammalian cell extracts and investigate how local translation impacts nascent protein localization and function. By monitoring the translation of the magnetically localized mRNAs, we show that mRNA-NP complexes operate as a source for the continuous production of proteins from defined positions. By applying this approach to actin-binding proteins, we triggered the local formation of actin cytoskeletons and identified the minimal requirements for spatial control of the actin filament network. In addition, our bottom-up approach identified a role for mRNA as a translation-coupled scaffold for the function of nascent N-terminal protein domains. Our approach will serve as a platform for regulating mRNA localization and investigating the function of nascent protein domains during translation.

Synthetic mRNA devices that detect endogenous proteins and distinguish mammalian cells.

Synthetic biology has great potential for future therapeutic applications including autonomous cell programming through the detection of protein signals and the production of desired outputs. Synthetic RNA devices are promising for this purpose. However, the number of available devices is limited due to the difficulty in the detection of endogenous proteins within a cell. Here, we show a strategy to construct synthetic mRNA devices that detect endogenous proteins in living cells, control translation and distinguish cell types. We engineered protein-binding aptamers that have increased stability in the secondary structures of their active conformation. The designed devices can efficiently respond to target proteins including human LIN28A and U1A proteins, while the original aptamers failed to do so. Moreover, mRNA delivery of an LIN28A-responsive device into human induced pluripotent stem cells (hiPSCs) revealed that we can distinguish living hiPSCs and differentiated cells by quantifying endogenous LIN28A protein expression level. Thus, our endogenous protein-driven RNA devices determine live-cell states and program mammalian cells based on intracellular protein information.

Cell-type-specific genome editing with a microRNA-responsive CRISPR-Cas9 switch.

The CRISPR-Cas9 system is a powerful genome-editing tool useful in a variety of biotechnology and biomedical applications. Here we developed a synthetic RNA-based, microRNA (miRNA)-responsive CRISPR-Cas9 system (miR-Cas9 switch) in which the genome editing activity of Cas9 can be modulated through endogenous miRNA signatures in mammalian cells. We created miR-Cas9 switches by using a miRNA-complementary sequence in the 5΄-UTR of mRNA encoding Streptococcus pyogenes Cas9. The miR-21-Cas9 or miR-302-Cas9 switches selectively and efficiently responded to miR-21-5p in HeLa cells or miR-302a-5p in human induced pluripotent stem cells, and post-transcriptionally attenuated the Cas9 activity only in the target cells. Moreover, the miR-Cas9 switches could differentially control the genome editing by sensing endogenous miRNA activities within a heterogeneous cell population. Our miR-Cas9 switch system provides a promising framework for cell-type selective genome editing and cell engineering based on intracellular miRNA information.

Competition between translation initiation factor eIF5 and its mimic protein 5MP determines non-AUG initiation rate genome-wide.

In the human genome, translation initiation from non-AUG codons plays an important role in various gene regulation programs. However, mechanisms regulating the non-AUG initiation rate remain poorly understood. Here, we show that the non-AUG initiation rate is nearly consistent under a fixed nucleotide context in various human and insect cells. Yet, it ranges from <1% to nearly 100% compared to AUG translation, depending on surrounding sequences, including Kozak, and possibly additional nucleotide contexts. Mechanistically, this range of non-AUG initiation is controlled in part, by the eIF5-mimic protein (5MP). 5MP represses non-AUG translation by competing with eIF5 for the Met-tRNAi-binding factor eIF2. Consistently, eIF5 increases, whereas 5MP decreases translation of NAT1/EIF4G2/DAP5, whose sole start codon is GUG. By modulating eIF5 and 5MP1 expression in combination with ribosome profiling we identified a handful of previously unknown non-AUG initiation sites, some of which serve as the exclusive start codons. If the initiation rate for these codons is low, then an AUG-initiated downstream ORF prevents the generation of shorter, AUG-initiated isoforms. We propose that the homeostasis of the non-AUG translatome is maintained through balanced expression of eIF5 and 5MP.

Efficient mRNA delivery system utilizing chimeric VSVG-L7Ae virus-like particles.

The delivery of mRNA is advantageous over DNA delivery as it is transient and does not carry the risk of genomic DNA integration. However, there are currently few efficient mRNA delivery options available, especially for hard-to-transfect cell types, and thus new delivery methods are needed. To this end, we have established a novel mRNA delivery system utilizing chimeric virus-like particles (VLPs). We generated a novel VLP by fusing protein G of Vesicular stomatitis virus (VSV-G) with a ribosomal protein L7Ae of Archeoglobus fulgidus. This system allowed the efficient delivery of EGFP mRNA which was independent from the presence of BoxC/D motif in the mRNA sequence. Our VSVG-L7Ae VLP system demonstrated high transduction efficacy in hard-to-transfect cell lines, such as human induced pluripotent stem cells (iPS cells) and monocytes. In summary, this platform may serve as an efficient and transient transgene delivery tool for an mRNA of interest.

RNA-based gene circuits for cell regulation.

A major goal of synthetic biology is to control cell behavior. RNA-mediated genetic switches (RNA switches) are devices that serve this purpose, as they can control gene expressions in response to input signals. In general, RNA switches consist of two domains: an aptamer domain, which binds to an input molecule, and an actuator domain, which controls the gene expression. An input binding to the aptamer can cause the actuator to alter the RNA structure, thus changing access to translation machinery. The assembly of multiple RNA switches has led to complex gene circuits for cell therapies, including the selective killing of pathological cells and purification of cell populations. The inclusion of RNA binding proteins, such as L7Ae, increases the repertoire and precision of the circuit. In this short review, we discuss synthetic RNA switches for gene regulation and their potential therapeutic applications.

Droplet-Shooting and Size-Filtration (DSSF) Method for Synthesis of Cell-Sized Liposomes with Controlled Lipid Compositions.

We report a centrifugal microfluidic method, droplet-shooting and size-filtration (DSSF), for the production of cell-sized liposomes with controlled lipid compositions. This involves the generation of large and small droplets from the tip of a glass capillary and the selective transfer of small droplets through an oil-water interface, thus resulting in the generation of cell-sized liposomes. We demonstrate control of the microdomain formation as well as the formation of asymmetric lipid bilayer liposomes of uniform size by the control of lipid composition. The DSSF method involves simple microfluidics and is easy to use. In addition, only a small volume (0.5-2 µL) of sample solution is required for the formation of hundreds of cell-sized liposomes. We believe that this method can be applied to generate cell-sized liposomes for a wide variety of uses, such as the construction of artificial cell-like systems.

Molecular robots with sensors and intelligence.

CONSPECTUS: What we can call a molecular robot is a set of molecular devices such as sensors, logic gates, and actuators integrated into a consistent system. The molecular robot is supposed to react autonomously to its environment by receiving molecular signals and making decisions by molecular computation. Building such a system has long been a dream of scientists; however, despite extensive efforts, systems having all three functions (sensing, computation, and actuation) have not been realized yet. This Account introduces an ongoing research project that focuses on the development of molecular robotics funded by MEXT (Ministry of Education, Culture, Sports, Science and Technology, Japan). This 5 year project started in July 2012 and is titled "Development of Molecular Robots Equipped with Sensors and Intelligence". The major issues in the field of molecular robotics all correspond to a feedback (i.e., plan-do-see) cycle of a robotic system. More specifically, these issues are (1) developing molecular sensors capable of handling a wide array of signals, (2) developing amplification methods of signals to drive molecular computing devices, (3) accelerating molecular computing, (4) developing actuators that are controllable by molecular computers, and (5) providing bodies of molecular robots encapsulating the above molecular devices, which implement the conformational changes and locomotion of the robots. In this Account, the latest contributions to the project are reported. There are four research teams in the project that specialize on sensing, intelligence, amoeba-like actuation, and slime-like actuation, respectively. The molecular sensor team is focusing on the development of molecular sensors that can handle a variety of signals. This team is also investigating methods to amplify signals from the molecular sensors. The molecular intelligence team is developing molecular computers and is currently focusing on a new photochemical technology for accelerating DNA-based computations. They also introduce novel computational models behind various kinds of molecular computers necessary for designing such computers. The amoeba robot team aims at constructing amoeba-like robots. The team is trying to incorporate motor proteins, including kinesin and microtubules (MTs), for use as actuators implemented in a liposomal compartment as a robot body. They are also developing a methodology to link DNA-based computation and molecular motor control. The slime robot team focuses on the development of slime-like robots. The team is evaluating various gels, including DNA gel and BZ gel, for use as actuators, as well as the body material to disperse various molecular devices in it. They also try to control the gel actuators by DNA signals coming from molecular computers.

Quantitative and simultaneous translational control of distinct mammalian mRNAs.

The introduction of multiple genes into cells is increasingly required for understanding and engineering biological systems. Small-molecule-responsive transcriptional regulation has been widely used to control transgene expression. In contrast, methods for specific and simultaneous regulation of multiple genes with a single regulatory protein remain undeveloped. In this report, we describe a method for quantitatively tuning the expression of multiple transgenes with a translational regulatory protein. A protein that binds a specific RNA motif inserted in the 5'-untranslated region (UTR) of an mRNA modulates the translation of that message in mammalian cells. We provide two independent mechanisms by which to rationally fine-tune the output: the efficiency of translation correlates well with the distance between the inserted motif and the 5' terminus of the mRNA and is further modulated by the tandem insertion of multiple RNA motifs. The combination of these two approaches allowed us to fine-tune the translational efficiency of target mRNAs over a wide dynamic range. Moreover, we controlled the expression of two transgenes simultaneously and specifically by engineering each cis-regulatory 5'-UTR. The approach provides a useful alternative regulatory layer for controlling gene expression in biological research and engineering.

Three-dimensionally designed protein-responsive RNA devices for cell signaling regulation.

The three-dimensional (3D) structures of many biomacromolecules have been solved to reveal the functions of these molecules. However, these 3D structures have rarely been applied to constructing efficient molecular devices that function in living cells. Here, we demonstrate a 3D structure-based molecular design principle for constructing short hairpin RNA (shRNA)-mediated genetic information converters; these converters respond to specific proteins and trigger the desired gene expression by modulating the function of the RNA-processing enzyme Dicer. The inhibitory effect on Dicer cleavage against the shRNA designed to specifically bind to U1A spliceosomal protein was correlated with the degree of steric hindrance between Dicer and the shRNA-protein complex in vitro: The level of the hindrance was predicted based on the models. Moreover, the regulation of gene expression was achieved by using the shRNA converters designed to bind to the target U1A or nuclear factor-κB (NF-κB) p50 proteins expressed in human cells. The 3D molecular design approach is widely applicable for developing new devices in synthetic biology.

RNA Switches Using Cas Proteins.

Expanding the number of available RNA-binding proteins (RBPs) is vital to establishing posttranscriptional circuits in mammalian cells. We focused on CRISPR-Cas systems and exploited Cas proteins for their versatility as RBPs. The translation of genes encoded in an mRNA becomes regulatable by a Cas protein by inserting a crRNA/sgRNA sequence recognizable by the specific Cas protein into its 5'UTR. These Cas protein-responsive switches vastly expand the available tools in synthetic biology because of the wide range of Cas protein orthologs that can be used as trigger proteins.Here, we describe the design principle of Cas protein-responsive switches, both plasmid and RNA versions, using Streptococcus pyogenes Cas9 (SpCas9) as an example and show an example of its use in mammalian cells, HEK293FT cells.

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.

Large-scale analysis of small molecule-RNA interactions using multiplexed RNA structure libraries.

The large-scale analysis of small-molecule binding to diverse RNA structures is key to understanding the required interaction properties and selectivity for developing RNA-binding molecules toward RNA-targeted therapies. Here, we report a new system for performing the large-scale analysis of small molecule-RNA interactions using a multiplexed pull-down assay with RNA structure libraries. The system profiled the RNA-binding landscapes of G-clamp and thiazole orange derivatives, which recognizes an unpaired guanine base and are good probes for fluorescent indicator displacement (FID) assays, respectively. We discuss the binding preferences of these molecules based on their large-scale affinity profiles. In addition, we selected combinations of fluorescent indicators and different ranks of RNA based on the information and screened for RNA-binding molecules using FID. RNAs with high- and intermediate-rank RNA provided reliable results. Our system provides fundamental information about small molecule-RNA interactions and facilitates the discovery of novel RNA-binding molecules.