Nucleic acid specific, photoswitchable fluorophores for monitoring RNAs in live cells

用于监测活细胞中 RNA 的核酸特异性光开关荧光团

• 批准号: 278738575
• 负责人: Professor Dr. Andriy Mokhir
• 金额: --
• 依托单位: Professur für Organische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/278738575?language=en
• 关键词: Nucleic; acid; specific; photoswitchable; fluorophores

Photochemical, nucleic acid-dependent (templated) reactions can potentially be applied in biological research to study endogenous ribonucleic acids in live cells with spatial and temporal resolution. For example, photoreduction of arylazide-based immolative linkers in the presence of Ru(II)-bipyridine complexes upon irradiation with 455 nm was applied to detect miR-21 in BT474 cells. Few other templated reactions have been reported, which are controlled by UV-light and short-wavelength visible light not exceeding 532 nm. However, such triggers are not ideally suitable for cellular applications. In particular, UV-light causes DNA mutations and is cytotoxic, whereas < 550 nm - visible light is efficiently absorbed by endogeneous riboflavins, thereby generating their excited triplet state. The latter state is quenched in cells with formation of singlet oxygen, superoxide anion radical and other reactive species, which induce DNA mutations, lead to depletion of riboflavins and damage of other biomolecules. These side effects limit cellular applications of photochemical templated reactions. In contrast, since cells are practically insensitive to light at wavelengths exceeding 650 nm, reagents responsive to this trigger are expected to be well compatible with live cells. The objective of this project is to develop caged fluorophores, which are activated by non-toxic red light in the presence of specific ribonucleic acids in live cells. All required components of the system including a caged fluorophore (substrate) and a photocatalyst, will be assembled on the RNA target due to highly specific hybridization reaction. Exposure of this associate to red light will lead to generation of singlet oxygen on the catalyst in close proximity to the substrate. The latter moiety will react with singlet oxygen leading to generation of a bright and photostable fluorophore, which can generate over 1000 photons upon its excitation. This great amplification effect will allow detecting a single dye by using fluorescence microscopy or single molecule fluorescence imaging. Based on this reaction a method for detection, localization and monitoring of rare endogenous RNAs (down to one sequence per cell) in live cells will be developed. The photocatalyst moiety will be optimized in such a way that it generates sufficient amount of the mediator to fully activate the caged fluorophore. However, after this desired reaction is complete, the photocatalyst will be quickly bleached thereby limiting the possible toxic effect of singlet oxygen.

光化学、核酸依赖性（模板化）反应可潜在地应用于生物学研究中，以空间和时间分辨率研究活细胞中的内源核糖核酸。例如，在 Ru(II)-联吡啶复合物存在下，在 455 nm 照射下，基于芳基叠氮化物的牺牲连接体发生光还原，用于检测 BT474 细胞中的 miR-21。很少有其他模板反应被报道，这些反应是由紫外光和不超过532 nm的短波长可见光控制的。然而，此类触发器并不非常适合蜂窝应用。特别是，紫外线会导致 DNA 突变并具有细胞毒性，而 < 550 nm - 可见光可被内源性核黄素有效吸收，从而产生其激发的三重态。后一种状态在细胞中被淬灭，形成单线态氧、超氧阴离子自由基和其他活性物质，从而诱导 DNA 突变，导致核黄素的消耗和其他生物分子的损伤。这些副作用限制了光化学模板反应的细胞应用。相比之下，由于细胞实际上对波长超过 650 nm 的光不敏感，因此响应这种触发的试剂预计与活细胞具有良好的相容性。该项目的目标是开发笼式荧光团，当活细胞中存在特定核糖核酸时，这种荧光团会被无毒的红光激活。由于高度特异性的杂交反应，系统所需的所有组件，包括笼式荧光团（底物）和光催化剂，都将组装在 RNA 靶标上。将该缔合物暴露于红光下将导致在靠近基材的催化剂上产生单线态氧。后一个部分将与单线态氧反应，产生明亮且光稳定的荧光团，在激发时可产生超过 1000 个光子。这种巨大的放大效应将允许使用荧光显微镜或单分子荧光成像来检测单一染料。基于该反应，将开发一种检测、定位和监测活细胞中稀有内源 RNA（每个细胞只有一个序列）的方法。光催化剂部分将以这样的方式进行优化，使其产生足够量的介体以完全激活笼状荧光团。然而，在这个所需的反应完成后，光催化剂将很快被漂白，从而限制单线态氧可能的毒性作用。

项目成果

Hybrids of a 9-anthracenyl moiety and fluorescein as chemodosimeters for the detection of singlet oxygen in live cells.

• DOI: 10.1039/c9ob02070e
• 发表时间: 2019-11
• 期刊: Organic & biomolecular chemistry
• 影响因子: 3.2
• 作者: S. Chercheja;S. Daum;Hong‐Gui Xu;Frank R. Beierlein;A. Mokhir

Cu2+-complexes as quenchers of photocatalytic activity of visible light-absorbing photosensitizers: An application in detection of nucleic acids

Cu2+配合物作为可见光吸收光敏剂光催化活性的猝灭剂：在核酸检测中的应用

• DOI: 10.1016/j.ica.2016.04.023
• 发表时间: 2016
• 期刊: Inorganica Chimica Acta
• 影响因子: 2.8
• 作者: Schikora;Mokhir
• 通讯作者: Mokhir

Endoperoxides Revealed as Origin of the Toxicity of Graphene Oxide.

• DOI: 10.1002/anie.201507070
• 发表时间: 2016-01
• 期刊: Angewandte Chemie
• 作者: H. Pieper;S. Chercheja;Siegfried Eigler;Christian E Halbig;M. Filipović;A. Mokhir

Red Light Triggered Fluorogenic Reaction with Picomolar Sensitivity Towards Nucleic Acids.

红光引发的荧光反应，对核酸具有皮摩尔灵敏度

• DOI: 10.1021/acs.bioconjchem.9b00299
• 发表时间: 2023
• 期刊: Bioconjugate chemistry
• 影响因子: 4.7
• 作者: Zozulia;Bachmann;Mokhir
• 通讯作者: Mokhir

[1,10]Phenanthroline based cyanine dyes as fluorescent probes for ribonucleic acids in live cells

• DOI: 10.1088/2050-6120/aa8510
• 发表时间: 2017-10
• 期刊: Methods and Applications in Fluorescence
• 影响因子: 3.2
• 作者: V. Kovalska;Marina V. Kuperman;O. Varzatskii;D. Kryvorotenko;E. Kinski;Margot Schikora;C. Janko;C. Alexiou;S. Yarmoluk;A. Mokhir