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

Biochemical and biomechanical drivers of cancer cell metastasis, drug response and nanomedicine

Metastasis, drug resistance and recurrence in cancer are regulated by the tumor microenvironment. This review describes recent advances in understanding how cancel cells respond to extracellular environmental cues via integrins, how to build engineered microenvironments to study these interactions in vitro and how nanomaterials can be used to detect and target tumor microenvironments

自己組織化ナノ集合体を利用した機能性バイオマテリアル開発

京都大学0048新制・課程博士博士(工学)甲第18595号工博第3956号新制||工||1608(附属図書館)31495京都大学大学院工学研究科合成・生物化学専攻(主査)教授 濵地 格, 教授 松田 建児, 教授 秋吉 一成学位規則第4条第1項該当Doctor of Philosophy (Engineering)Kyoto UniversityDFA

Golgi Recruitment Assay for Visualizing Small-Molecule Ligand–target Engagement in Cells

The development of methods that allow detection of ligand–target engagement in cells is an important challenge in chemical biology and drug discovery. Here, we present a Golgi recruitment (G-REC) assay in which the ligand binding to the target protein can be visualized as Golgi-localized fluorescence signals. We show that the G-REC assay is applicable to the detection of various ligand–target interactions, ligand affinity comparison among distinct protein isoforms, and the monitoring of unmodified drug–target engagement in cells.</b

Chemically Reactive Supramolecular Hydrogel Coupled with a Signal Amplification System for Enhanced Analyte Sensitivity

Multicomponent supramolecular hydrogels are constructed for sensitive, naked-eye detection of small-molecule biomarkers. A dendritic self-immolative molecule and the corresponding enzyme as a signal amplification system were stably embedded in a hydrogen peroxide (H₂O₂)-responsive supramolecular hydrogel (<b>BPmoc-F</b>_<b>3</b>), together with other enzymes. The nanostructure and mechanical strength of the hybrid <b>BPmoc-F</b>_<b>3</b> gel were not substantially diminished by incorporation of these multiple components in the absence of target biomarkers, but could be destroyed by addition of the biomarker through the multiple enzymatic and chemical cascade reactions operating in combination within the gel matrix. The sensitivity to biomarkers such as H₂O₂, glucose, and uric acid, detected by gel–sol transition, was significantly enhanced by the signal amplification system. An array chip consisting of these multicomponent hydrogels enabled the detection of the level of hyperuricemia disease in human plasma samples

Chemogenetic Control of Protein Anchoring to Endomembranes in Living Cells with Lipid-Tethered Small Molecules

The Self-localizing Ligand-Induced Protein Translocation(SLIPT) system is an emerging platform that controls protein localization in living cells using synthetic self-localizing ligands (SLs). Here, we report a chemogenetic SLIPT system for inducing protein translocation from the cytoplasm to the surface of the endoplasmic reticulum (ER) and Golgi membranes, referred to as endomembranes.By screening a series of lipid-trimethoprim (TMP) conjugates, we found oleic acid-tethered TMP (oleTMP) to be the optimal SL that efficiently relocated and anchored Escherichiacoli dihydrofolate reductase (eDHFR)-fusion proteins toendomembranes. We showed that oleTMP mediated protein anchoring to endomembranes within minutes and could be reversed by the addition of free TMP. We also applied the endomembrane SLIPT system to artificially activate endomembrane Ras and inhibit the active nuclear transport of extracellular signal-regulated kinase (ERK), demonstrating its applicability for manipulating biological processes in living cells. We envision that the present oleTMP-based SLIPT system, which affords rapid and reversible control of protein anchoring to endomembranes, will offer a new unique tool for the study and control of spatiotemporally regulated cell signaling processes

Intracellular Protein-Responsive Supramolecules: Protein Sensing and In-Cell Construction of Inhibitor Assay System

Supramolecular nanomaterials responsive to specific intracellular proteins should be greatly promising for protein sensing and imaging, controlled drug release or dynamic regulation of cellular processes. However, valid design strategies to create useful probes are poorly developed, particularly for proteins inside living cells as targets. We recently reported a unique supramolecular strategy for specific protein detection using self-assembling fluorescent probes consisting of a protein ligand and a fluorophore on the live cell surface, as well as in test tube settings. Herein, we discovered that our self-assembled supramolecular probes having a rhodamine derivative (tetramethylrhodamine or rhodamine-green) can incorporate and stay as less-fluorescent aggregates inside the living cells, so as to sense the protein activity in a reversible manner. Using the overexpressed model protein (dihydrofolate reductase), we demonstrated that this turn-on/off mode is controlled by selective ligand–protein recognition inside the live cells. Not only such a model protein, but also endogenous human carbonic anhydrase and heat shock protein 90 were specifically visualized in living mammalian cells, by use of the similar ligand-tethered supramolecular probes. Furthermore, such reversibility allowed us to intracellularly construct a unique system to evaluate the inhibitors affinity toward specific endogenous proteins in live cells, highlighting the potential of dynamic supramolecules as novel intelligent biomaterials