Altering the orientation of a fused protein to the RNA-binding ribosomal protein L7Ae and its derivatives through circular permutation.

RNA-protein complexes (RNPs) are useful for constructing functional nano-objects because a variety of functional proteins can be displayed on a designed RNA scaffold. Here, we report circular permutations of an RNA-binding protein L7Ae based on the three-dimensional structure information to alter the orientation of the displayed proteins on the RNA scaffold. An electrophoretic mobility shift assay and atomic force microscopy (AFM) analysis revealed that most of the designed circular permutants formed an RNP nano-object. Moreover, the alteration of the enhanced green fluorescent protein (EGFP) orientation was confirmed with AFM by employing EGFP on the L7Ae permutant on the RNA. The results demonstrate that targeted fine-tuning of the stereo-specific fixation of a protein on a protein-binding RNA is feasible by using the circular permutation technique.

A trifunctional, triangular RNA-protein complex.

Multifunctional molecular complexes are valuable tools with a variety of applications. We have developed an RNA-protein complex (RNP) containing three different proteins attached to the tips of a triangular RNA scaffold. We designed and constructed three RNA strands that specifically bind a ribosomal protein, L7Ae, and that autonomously form a single triangular RNP via RNA kissing loop (KL) interactions. This RNP-based approach can be used as an alternative tool to produce unique, multifunctional molecules with customized dimensions, functions, and targets.

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.

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.

A versatile cis-acting inverter module for synthetic translational switches.

Artificial genetic switches have been designed and tuned individually in living cells. A method to directly invert an existing OFF switch to an ON switch should be highly convenient to construct complex circuits from well-characterized modules, but developing such a technique has remained a challenge. Here we present a cis-acting RNA module to invert the function of a synthetic translational OFF switch to an ON switch in mammalian cells. This inversion maintains the property of the parental switch in response to a particular input signal. In addition, we demonstrate simultaneous and specific expression control of both the OFF and ON switches. The module fits the criteria of universality and expands the versatility of mRNA-based information processing systems developed for artificially controlling mammalian cellular behaviour.

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.

Directed evolution of a synthetic RNA-protein module to create a new translational switch.

A synthetic RNP-binding module was developed to produce an alternative translational switch: an in vitro selection experiment targeting a derivative of L7Ae protein (L7KK) identified a new H23 RNA aptamer that binds tightly to both L7KK and L7Ae. The switch serves as a translational OFF switch for constructing a NOR gate.

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.

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.

Self-replication reactions dependent on tertiary interaction motifs in an RNA ligase ribozyme.

RNA can function both as an informational molecule and as a catalyst in living organisms. This duality is the premise of the RNA world hypothesis. However, one flaw in the hypothesis that RNA was the most essential molecule in primitive life is that no RNA self-replicating system has been found in nature. To verify whether RNA has the potential for self-replication, we constructed a new RNA self-assembling ribozyme that could have conducted an evolvable RNA self-replication reaction. The artificially designed, in vitro selected ligase ribozyme was employed as a prototype for a self-assembling ribozyme. The ribozyme is composed of two RNA fragments (form R1·Z1) that recognize another R1·Z1 molecule as their substrate and perform the high turnover ligation reaction via two RNA tertiary interaction motifs. Furthermore, the substrate recognition of R1·Z1 is tolerant of mutations, generating diversity in the corresponding RNA self-replicating network. Thus, we propose that our system implies the significance of RNA tertiary motifs in the early RNA molecular evolution of the RNA world.

Light-regulated mRNA condensation by a photosensitive surfactant works as a series photoswitch of translation activity in the presence of small RNAs.

AzoTAB, a photosensitive azobenzene cationic surfactant, which phototriggers translation activity through light-regulated condensation of mRNA, is added to a translation solution containing several mRNAs, which can be selectively silenced by specific small RNAs. We find that gene silencing by small RNAs remains functional regardless of AzoTAB concentration and UV illumination. In the absence of UV, the translation of all genes present in the medium is partially to fully inhibited depending on AzoTAB concentration. In contrast, the application of a short UV stimulus (365 nm for 1.5 min) results in the selective photoactivation of genes that are not silenced by small RNA. These results show that light-regulated condensation by AzoTAB works as a sequence-independent series photoswitch added to parallel sequence-specific regulation by small RNAs.

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 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.

Rational optimization of the DSL ligase ribozyme with GNRA/receptor interacting modules.

The DSL ribozyme is a class of artificial ligase ribozymes with a highly modular architecture, which catalyzes template-directed RNA ligation on a helical substrate module that can be either covalently connected (cis-DSL) or physically separated (trans-DSL) from the catalytic module. Substrate recognition by the catalytic module is promoted by one or two sets of GNRA/receptor interactions acting as clamps in the cis or trans configurations, respectively. In this study, we have rationally designed and analyzed the catalytic and self-assembly properties of several trans-DSL ribozymes with different sets of natural and artificial GNRA-receptor clamps. Two variants newly designed in this study showed significantly enhanced catalytic properties with respect of the original trans-DSL construct. While this work allows dissection of the turnover and catalytic properties of the trans-DSL ribozyme, it also emphasizes the remarkable modularity of RNA tertiary structure for nano-construction of complex functions.

Coordinated control of a designed trans-acting ligase ribozyme by a loop-receptor interaction.

We previously developed a synthetic cis-acting RNA ligase ribozyme with 3'-5' joining activity termed "DSL" (designed and selected ligase). DSL was easily transformed into a trans-acting form because of its highly modular architecture. In this study, we investigated the modular properties and turnover capabilities of a trans-acting DSL, tDSL-1/GUAA. tDSL-1/GUAA exhibited remarkably high activity compared with the parental cis-acting DSL, and it attained a high turnover number. Taken together, the results indicate that a loop-receptor interaction plays a significant role in determining the activity of the trans-acting ribozyme and in its ability to perform multiple turnovers of the reaction.

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.

A designed RNA selection: establishment of a stable complex between a target and selectant RNA via two coordinated interactions.

In this paper, we describe a new method for selecting RNA aptamers that cooperatively bind to two specific sites within a target RNA. We designed a selection system in which two RNAs, a target RNA and a RNA pool, were assembled by employing a pre-organized GAAA tetraloop-11-nt receptor interaction. This allows us to select the binding sequence against a targeted internal loop as well as a linker region optimized for binding of the two binding sites. After the selection, the aptamers bound with dissociation constants in the nanomolar range, thereby forming a stable complex with the target RNA. Thus this method enables identification of aptamers for a specific binding site together with a linker for cooperative binding of the two RNAs. It appears that our new method can be applied generally to select RNAs that adhere tightly to a target RNA via two specific sites. The method can also be applicable for further engineering of both natural and artificial RNAs.

Concerted effects of two activator modules on the group I ribozyme reaction.

Group I intron ribozymes have a modular architecture and structural elements essential for catalysis. The elements are located in the conserved modular domain P3-P7 that is stabilized by another conserved module, P4-P6. It has been reported that artificial modules can complement the function of the native P4-P6. To exploit the modular architecture of group I ribozyme, we have constructed a hybrid ribozyme by attaching an artificial activator module to the wild-type T4 td ribozyme. Kinetic analysis of the hybrid ribozyme revealed that the artificial module and P4-P6 have unusual positive and negative concerted effects in activating the ribozyme.