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

光化学、核酸依赖(模板化)反应可潜在地应用于生物学研究，以空间和时间分辨率研究活细胞中的内源性核糖核酸。例如，在Ru(II)-联吡啶配合物存在的情况下，在455 nm的光照射下，芳拉齐特基自燃连接体被应用于检测BT474细胞中的miR-21。几乎没有报道其他模板化反应，这些反应是由紫外光和不超过532 nm的短波可见光控制的。然而，这样的触发器并不理想地适用于蜂窝应用。特别是，紫外光会导致DNA突变并具有细胞毒性，而550 nm-可见光可以有效地被内源性核黄素吸收，从而产生它们的激发三线态。后者在细胞内猝灭，形成单线态氧、超氧阴离子自由基和其他活性物种，导致DNA突变，导致核黄素耗竭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在细胞中的应用。相反，由于细胞对波长超过650纳米的光几乎不敏感，因此对这种触发反应的试剂有望与活细胞很好地兼容。这个项目的目标是开发笼式荧光团，在活细胞中存在特定的核糖核酸的情况下，通过无毒的红光激活这种荧光团。由于高度特异的杂交反应，该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。将这种缔合物暴露在红光下将导致催化剂上靠近底物的单线态氧的产生。后者与单线态氧反应生成明亮的、光稳定的荧光团，激发后可产生1000多个光子。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的染料。基于这一反应，将发展一种检测、定位和监测活细胞中稀有内源RNA(低至每个细胞一个序列)的方法。光催化剂部分将被优化，以产生足够数量的介体来完全激活笼子中的荧光团。然而，在这个所需的反应完成后，光催化剂将被快速漂白，从而限制了单线态氧可能产生的毒性效应。

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.