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

光化学，核酸依赖性（模板）反应可以潜在地应用于生物学研究，以研究活细胞中的内源性核糖核酸的空间和时间分辨率。例如，在Ru（II）-联吡啶络合物存在下，在用455 nm照射时，基于芳基叠氮化物的牺牲性接头的光还原被应用于检测BT474细胞中的miR-21。很少有其他模板反应被报道，这是由紫外光和短波长不超过532 nm的可见光控制。然而，这样的触发器并不理想地适合于蜂窝应用。特别地，UV光引起DNA突变并且是细胞毒性的，而< 550 nm -可见光被内源性核黄素有效地吸收，从而产生它们的激发三重态。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反应性物质，其诱导DNA突变，导致核黄素耗尽和其他生物分子损伤。这些副作用限制了光化学模板化反应的细胞应用。相反，由于细胞实际上对波长超过650 nm的光不敏感，因此预期响应于该触发的试剂与活细胞良好相容。该项目的目标是开发笼状荧光团，在活细胞中存在特定核糖核酸的情况下，这些荧光团被无毒红光激活。由于高度特异性的杂交反应，系统的所有所需组分，包括笼状荧光团（底物）和光催化剂，将组装在RNA靶标上。将该缔合物暴露于红光将导致在紧邻基底的催化剂上产生单线态氧。后一部分将与单线态氧反应，导致产生明亮且光稳定的荧光团，其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通过使用荧光显微镜或单分子荧光成像来检测单个染料。基于该反应，将开发用于检测、定位和监测活细胞中的罕见内源RNA（低至每个细胞一个序列）的方法。光催化剂部分将以这样的方式进行优化，使得其产生足够量的介体以完全激活笼状荧光团。然而，在该期望的反应完成之后，光催化剂将被快速漂白，从而限制单线态氧的可能的毒性作用。

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.