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

mRNA医薬の課題克服に向けた「環状RNAスイッチ」の開発 --RNAによる持続的な遺伝子発現と細胞種特異的な制御に成功--. 京都大学プレスリリース. 2023-01-16.Cyclic RNA Switches that Regulate Gene Expression in a Cell Type-Specific Manner. 京都大学プレスリリース. 2023-02-16.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

Target-dependent RNA polymerase as universal platform for gene expression control in response to intracellular molecules

抗体を用いた標的依存性RNAポリメラーゼの開発 多様な細胞内分子に応答する遺伝子発現制御のプラットフォーム. 京都大学プレスリリース. 2023-11-17.Using Single-antibodies as a New Ingenious Tool to Build Bio-circuitry. 京都大学プレスリリース. 2023-11-17.Controlling gene expression in response to specific molecules is an essential technique for regulating cellular functions. However, current platforms with transcription and translation regulators have a limited number of detectable molecules to induce gene expression. Here to address these issues, we present a Target-dependent RNA polymerase (TdRNAP) that can induce RNA transcription in response to the intracellular target specifically recognized by single antibody. By substituting the fused antibody, we demonstrate that TdRNAPs respond to a wide variety of molecules, including peptides, proteins, RNA, and small molecules, and produce desired transcripts in human cells. Furthermore, we show that multiple TdRNAPs can construct orthogonal and multilayer genetic circuits. Finally, we apply TdRNAP to achieve cell-specific genome editing that is autonomously triggered by detecting the target gene product. TdRNAP can expand the molecular variety for controlling gene expression and provide the genetic toolbox for bioengineering and future therapeutic applications

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

様々な5'キャップ構造をもつ機能的なmRNAの汎用的な合成方法 酵素を用いて簡便かつ効率的に. 京都大学プレスリリース. 2023-02-03.Development of a versatile method to synthesize functional mRNAs with diverse 5' cap structures. 京都大学プレスリリース. 2023-02-03.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

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

Fe-K line probing of material around the AGN central engine with Suzaku

We systematically analyzed the high-quality Suzaku data of 88 Seyfert galaxies. We obtained a clear relation between the absorption column density and the equivalent width of the 6.4 keV line above 10$^{23}$ cm$^{-2}$, suggesting a wide-ranging column density of $10^{23-24.5}$ cm$^{-2}$ with a similar solid and a Fe abundance of 0.7--1.3 solar for Seyfert 2 galaxies. The EW of the 6.4 keV line for Seyfert 1 galaxies are typically 40--120 eV, suggesting the existence of Compton-thick matter like the torus with a column density of $>10^{23}$ cm$^{-2}$ and a solid angle of $(0.15-0.4)*4pi$, and no difference of neutral matter is visible between Seyfert 1 and 2 galaxies. An absorber with a lower column density of $10^{21-23}$ cm$^{-2}$ for Compton-thin Seyfert 2 galaxies is suggested to be not a torus but an interstellar medium. These constraints can be understood by the fact that the 6.4 keV line intensity ratio against the 10--50 keV flux is almost identical within a range of 2--3 in many Seyfert galaxies. Interestingly, objects exist with a low EW, 10--30 eV, of the 6.4 keV line, suggesting that those torus subtends only a small solid angle of $<0.2*4pi$. Ionized Fe-K$\alpha$ emission or absorption lines are detected from several percents of AGNs. Considering the ionization state and equivalent width, emitters and absorbers of ionized Fe-K lines can be explained by the same origin, and highly ionized matter is located at the broad line region. The rapid increase in EW of the ionized Fe-K emission lines at $N_{H}>10^{23}$ cm$^{-2}$ is found, like that of the cold material. It is found that these features seem to change for brighter objects with more than several $10^{44}$ erg/s such that the Fe-K line features become weak. We discuss this feature, together with the torus structure.Comment: 32 pages, 20 figures, ApJ accepte

Paracrine IL-33 Stimulation Enhances Lipopolysaccharide-Mediated Macrophage Activation

BACKGROUND: IL-33, a member of the IL-1 family of cytokines, provokes Th2-type inflammation accompanied by accumulation of eosinophils through IL-33R, which consists of ST2 and IL-1RAcP. We previously demonstrated that macrophages produce IL-33 in response to LPS. Some immune responses were shown to differ between ST2-deficient mice and soluble ST2-Fc fusion protein-treated mice. Even in anti-ST2 antibody (Ab)-treated mice, the phenotypes differed between distinct Ab clones, because the characterization of such Abs (i.e., depletion, agonistic or blocking Abs) was unclear in some cases. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate the precise role of IL-33, we newly generated neutralizing monoclonal Abs for IL-33. Exogenous IL-33 potentiated LPS-mediated cytokine production by macrophages. That LPS-mediated cytokine production by macrophages was suppressed by inhibition of endogenous IL-33 by the anti-IL-33 neutralizing mAbs. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that LPS-mediated macrophage activation is accelerated by macrophage-derived paracrine IL-33 stimulation

Investigation of hitting ability on distance in developing player

J-GLOBAL ID : 200901006236324733This study was examined the relationship between batting ability and distance in baseball developing players. The results revealed that elementary school students use launch angle to produce distance. It was also revealed that high school students hitting tactically with exit velocity and launch angle. It was thought that elementary school students produce distance by raising launch angle. It was suggested that as their exit velocity improved with growth, they were hitting for extra-base hits while adjusting launch angle. The relationship between BMI and hitting ability, it was considered that bat speed and exit velocity increase from junior high school onward due to increased muscle mass and improved swing technique, leading to extra-base hits. These results revealed the characteristics of batting performance by age group. This may have provided knowledge into the coaching of hitting that leads to extra-base hits and on base.1520860078892890368application/pdfdepartmental bulletin pape

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