細胞内分子に応答した遺伝子発現制御を実現する標的依存性RNAポリメラーゼの開発

京都大学新制・課程博士博士(医科学)甲第25206号医科博第162号京都大学大学院医学研究科医科学専攻(主査)教授 遊佐 宏介, 教授 竹内 理, 教授 近藤 玄学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDFA

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

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

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.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

Synthesis of practical red fluorescent probe for cytoplasmic calcium ions with greatly improved cell-membrane permeability

In this data article, we described the detailed synthetic procedure and the experimental data for the synthesis of a red-fluorescent probe for calcium ions (Ca2+) with improved water solubility. This Ca2+ red-fluorescent probe CaTM-3 AM could be applied to fluorescence imaging of physiological Ca2+ concentration changes in not only live cells, but also brain slices, with high cell-membrane permeability leading to bright fluorescence in biosamples. The data provided herein are in association with the research article “The Development of Practical Red Fluorescent Probe for Cytoplasmic Calcium Ions with Greatly Improved Cell-membrane Permeability” in Cell Calcium (Hirabayashi et al., 2016) [1]

Reversible Off–On Fluorescence Probe for Hypoxia and Imaging of Hypoxia–Normoxia Cycles in Live Cells

We report a fully reversible off–on fluorescence probe for hypoxia. The design employs QSY-21 as a Förster resonance energy transfer (FRET) acceptor and cyanine dye Cy5 as a FRET donor, based on our finding that QSY-21 undergoes one-electron bioreduction to the radical under hypoxia, with an absorbance decrease at 660 nm. At that point, FRET can no longer occur, and the dye becomes strongly fluorescent. Upon recovery of normoxia, the radical is immediately reoxidized to QSY-21, with loss of fluorescence due to restoration of FRET. We show that this probe, RHyCy5, can monitor repeated hypoxia–normoxia cycles in live cells

Development of a Series of Practical Fluorescent Chemical Tools To Measure pH Values in Living Samples

In biological systems, the pH in intracellular organelles or tissues is strictly regulated, and differences of pH are deeply related to key biological events such as protein degradation, intracellular trafficking, renal failure, and cancer. Ratiometric fluorescence imaging is useful for determination of precise pH values, but existing fluorescence probes have substantial limitations, such as inappropriate p<i>K</i>_a for imaging in the physiological pH range, inadequate photobleaching resistance, and insufficiently long excitation and emission wavelengths. Here we report a versatile scaffold for ratiometric fluorescence pH probes, based on asymmetric rhodamine. To demonstrate its usefulness for biological applications, we employed it to develop two probes. (1) <b>SiRpH5</b> has suitable p<i>K</i>_a and water solubility for imaging in acidic intracellular compartments; by using transferrin tagged with <b>SiRpH5</b>, we achieved time-lapse imaging of pH in endocytic compartments during protein trafficking for the first time. (2) <b>Me-pEPPR</b> is a near-infrared (NIR) probe; by using dextrin tagged with <b>Me-pEPPR</b>, we were able to image extracellular pH of renal tubules and tumors in situ. These chemical tools should be useful for studying the influence of intra- and extracellular pH on biological processes, as well as for in vivo imaging

Development of a Series of Practical Fluorescent Chemical Tools To Measure pH Values in Living Samples

In biological systems, the pH in intracellular organelles or tissues is strictly regulated, and differences of pH are deeply related to key biological events such as protein degradation, intracellular trafficking, renal failure, and cancer. Ratiometric fluorescence imaging is useful for determination of precise pH values, but existing fluorescence probes have substantial limitations, such as inappropriate p<i>K</i>_a for imaging in the physiological pH range, inadequate photobleaching resistance, and insufficiently long excitation and emission wavelengths. Here we report a versatile scaffold for ratiometric fluorescence pH probes, based on asymmetric rhodamine. To demonstrate its usefulness for biological applications, we employed it to develop two probes. (1) <b>SiRpH5</b> has suitable p<i>K</i>_a and water solubility for imaging in acidic intracellular compartments; by using transferrin tagged with <b>SiRpH5</b>, we achieved time-lapse imaging of pH in endocytic compartments during protein trafficking for the first time. (2) <b>Me-pEPPR</b> is a near-infrared (NIR) probe; by using dextrin tagged with <b>Me-pEPPR</b>, we were able to image extracellular pH of renal tubules and tumors in situ. These chemical tools should be useful for studying the influence of intra- and extracellular pH on biological processes, as well as for in vivo imaging

Development of a Series of Practical Fluorescent Chemical Tools To Measure pH Values in Living Samples

In biological systems, the pH in intracellular organelles or tissues is strictly regulated, and differences of pH are deeply related to key biological events such as protein degradation, intracellular trafficking, renal failure, and cancer. Ratiometric fluorescence imaging is useful for determination of precise pH values, but existing fluorescence probes have substantial limitations, such as inappropriate p<i>K</i>_a for imaging in the physiological pH range, inadequate photobleaching resistance, and insufficiently long excitation and emission wavelengths. Here we report a versatile scaffold for ratiometric fluorescence pH probes, based on asymmetric rhodamine. To demonstrate its usefulness for biological applications, we employed it to develop two probes. (1) <b>SiRpH5</b> has suitable p<i>K</i>_a and water solubility for imaging in acidic intracellular compartments; by using transferrin tagged with <b>SiRpH5</b>, we achieved time-lapse imaging of pH in endocytic compartments during protein trafficking for the first time. (2) <b>Me-pEPPR</b> is a near-infrared (NIR) probe; by using dextrin tagged with <b>Me-pEPPR</b>, we were able to image extracellular pH of renal tubules and tumors in situ. These chemical tools should be useful for studying the influence of intra- and extracellular pH on biological processes, as well as for in vivo imaging