High-resolution Identification and Separation of Living Cell Types by Multiple microRNA-responsive Synthetic mRNAs

合成RNAを利用した生細胞の高精度な同定と分離. 京都大学プレスリリース. 2016-02-25.The precise identification and separation of living cell types is critical to both study cell function and prepare cells for medical applications. However, intracellular information to distinguish live cells remains largely inaccessible. Here, we develop a method for high-resolution identification and separation of cell types by quantifying multiple microRNA (miRNA) activities in live cell populations. We found that a set of miRNA-responsive, in vitro synthesized mRNAs identify a specific cell population as a sharp peak and clearly separate different cell types based on less than two-fold differences in miRNA activities. Increasing the number of miRNA-responsive mRNAs enhanced the capability for cell identification and separation, as we precisely and simultaneously distinguished different cell types with similar miRNA profiles. In addition, the set of synthetic mRNAs separated HeLa cells into subgroups, uncovering heterogeneity of the cells and the level of resolution achievable. Our method could identify target live cells and improve the efficiency of cell purification from heterogeneous populations

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

Numerical operations in living cells by programmable RNA devices

細胞内の複数のマイクロRNAを同時に検知して細胞を生きたまま精密に分けることに成功. 京都大学プレスリリース. 2019-08-30.Integrated bioengineering systems can make executable decisions according to the cell state. To sense the state, multiple biomarkers are detected and processed via logic gates with synthetic biological devices. However, numerical operations have not been achieved. Here, we show a design principle for messenger RNA (mRNA) devices that recapitulates intracellular information by multivariate calculations in single living cells. On the basis of this principle and the collected profiles of multiple microRNA activities, we demonstrate that rationally programmed mRNA sets classify living human cells and track their change during differentiation. Our mRNA devices automatically perform multivariate calculation and function as a decision-maker in response to dynamic intracellular changes in living cells

Motif programming: a microgene-based method for creating synthetic proteins containing multiple functional motifs

The presence of peptide motifs within the proteins provides the synthetic biologist with the opportunity to fabricate novel proteins through the programming of these motifs. Here we describe a method that enables one to combine multiple peptide motifs to generate a combinatorial protein library. With this method, a set of sense and antisense oligonucleotide primers were prepared. These primers were mixed and polymerized, so that the resultant DNA consisted of combinatorial polymers of multiple microgenes created from the stochastic assembly of the sense and antisense primers. With this motif-mixing method, we prepared a protein library from the BH1-4 motifs shared among Bcl-2 family proteins. Among the 41 clones created, 70% of clones had a stable, presumably folded expression product in human cells, which was detectable by immunohistochemistry and western blot. The proteins obtained varied with respect to both the number and the order of the four motifs. The method enables homology-independent polymerization of DNA blocks that coded motif sequences, and the frequency of each motif within a library can be adjusted in a tailor-made manner. This motif programming has a potential for creating a library with a large proportion of folded/functional proteins

A protocol to construct RNA-protein devices for photochemical translational regulation of synthetic mRNAs in mammalian cells

Here, we describe a protocol for the translational regulation of transfected messenger RNAs (mRNAs) using light in mammalian cells. We detail the steps for photocaged ligand synthesis, template DNA preparation, and mRNA synthesis. We describe steps for mRNA transfection, treatment of cells with a photocaged ligand followed by light irradiation, and analysis of the transgene expression. The protocol enables spatiotemporally regulated transgene expression without the risk of insertional mutagenesis

RNA structure-wide discovery of functional interactions with multiplexed RNA motif library

RNA構造のライブラリ化を通じてRNA 構造ごとにおけるRNA-タンパク質相互作用を大規模に解析するシステム「FOREST」の開発 --RNAを標的とする創薬に道--. 京都大学プレスリリース. 2020-12-09.Biochemical assays and computational analyses have discovered RNA structures throughout various transcripts. However, the roles of these structures are mostly unknown. Here we develop folded RNA element profiling with structure library (FOREST), a multiplexed affinity assay system to identify functional interactions from transcriptome-wide RNA structure datasets. We generate an RNA structure library by extracting validated or predicted RNA motifs from gene-annotated RNA regions. The RNA structure library with an affinity enrichment assay allows for the comprehensive identification of target-binding RNA sequences and structures in a high-throughput manner. As a proof-of-concept, FOREST discovers multiple RNA-protein interaction networks with quantitative scores, including translational regulatory elements that function in living cells. Moreover, FOREST reveals different binding landscapes of RNA G-quadruplex (rG4) structures-binding proteins and discovers rG4 structures in the terminal loops of precursor microRNAs. Overall, FOREST serves as a versatile platform to investigate RNA structure-function relationships on a large scale

A versatile and robust cell purification system with an RNA-only circuit composed of microRNA-responsive ON and OFF switches

２つの合成mRNAスイッチを活用した純度の高い細胞選別システムの開発. 京都大学プレスリリース. 2022-01-06.Synthetic gene circuits that improve stem cell quality. 京都大学プレスリリース. 2022-01-06.Human induced pluripotent stem cells (iPSCs) are promising cell resources for cell therapy and drug discovery. However, iPSC-derived differentiated cells are often heterogenous and need purification using a flow cytometer, which has high cost and time consumption for large-scale purification. MicroRNAs (miRNAs) can be used as cell selection markers, because their activity differs between cell types. Here, we show miRNA-responsive ON and OFF switch mRNAs for robust cell purification. The ON switch contains a miRNA-target sequence after the polyadenylate tail, triggering translational activation by sensing the target miRNA. By designing RNA-only circuits with miRNA-ON and -OFF switch mRNAs that encode a lethal ribonuclease, Barnase, and its inhibitor, Barstar, we efficiently purified specific cell types, including human iPSCs and differentiated cardiomyocytes, without flow cytometry. Synthetic mRNA circuits composed of ON and OFF switches provide a safe, versatile, and time-saving method to purify various cell types for biological and clinical 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

Programmable mammalian translational modulators by CRISPR-associated proteins

mRNAスイッチを用いた哺乳類細胞内コンピューティングの基盤構築. 京都大学プレスリリース. 2023-04-20.Establishing the basis for mammalian intracellular computing with mRNA switches. 京都大学プレスリリース. 2023-04-21.Translational modulation based on RNA-binding proteins can be used to construct artificial gene circuits, but RNA-binding proteins capable of regulating translation efficiently and orthogonally remain scarce. Here we report CARTRIDGE (Cas-Responsive Translational Regulation Integratable into Diverse Gene control) to repurpose Cas proteins as translational modulators in mammalian cells. We demonstrate that a set of Cas proteins efficiently and orthogonally repress or activate the translation of designed mRNAs that contain a Cas-binding RNA motif in the 5’-UTR. By linking multiple Cas-mediated translational modulators, we designed and built artificial circuits like logic gates, cascades, and half-subtractor circuits. Moreover, we show that various CRISPR-related technologies like anti-CRISPR and split-Cas9 platforms could be similarly repurposed to control translation. Coupling Cas-mediated translational and transcriptional regulation enhanced the complexity of synthetic circuits built by only introducing a few additional elements. Collectively, CARTRIDGE has enormous potential as a versatile molecular toolkit for mammalian synthetic biology