Twisted Cyanines: A Non-Planar Fluorogenic Dye with Superior Photostability and its Use in a Protein-Based Fluoromodule

The cyanine dye thiazole orange (TO) is a well-known fluorogenic stain for DNA and RNA, but this property precludes its use as an intracellular fluorescent probe for non-nucleic acid biomolecules. Further, as is the case with many cyanines, the dye suffers from low photostability. Here, we report the synthesis of a bridge-substituted version of TO named α-CN-TO, where the central methine hydrogen of TO is replaced by an electron withdrawing cyano group, which was expected to decrease the susceptibility of the dye toward singlet oxygen-mediated degradation. An X-ray crystal structure shows that α-CN-TO is twisted drastically out of plane, in contrast to TO, which crystallizes in the planar conformation. α-CN-TO retains the fluorogenic behavior of the parent dye TO in viscous glycerol/water solvent, but direct irradiation and indirect bleaching studies showed that α-CN-TO is essentially inert to visible light and singlet oxygen. In addition, the twisted conformation of α-CN-TO mitigates nonspecific binding and fluorescence activation by DNA and a previously selected TO-binding protein and exhibits low background fluorescence in HeLa cell culture. α-CN-TO was then used to select a new protein that binds and activates fluorescence from the dye. The new α-CN-TO/protein fluoromodule exhibits superior photostability to an analogous TO/protein fluoromodule. These properties indicate that α-CN-TO will be a useful fluorogenic dye in combination with specific RNA and protein binding partners for both in vitro and cell-based applications. More broadly, structural features that promote nonplanar conformations can provide an effective method for reducing nonspecific binding of cationic dyes to nucleic acids and other biomolecules.噻唑橙（thiazole orange, TO）是一类广为人知的用于DNA和RNA标记的荧光生成型染色剂（fluorogenic stain），但该特性使其无法作为针对非核酸生物分子的细胞内荧光探针（intracellular fluorescent probe）使用。此外，与多数花菁染料（cyanine dye）类似，该染料的光稳定性（photostability）较差。我们在此报道了一种TO的桥位取代衍生物α-CN-TO的合成方法：将TO的中心次甲基氢（methine hydrogen）替换为吸电子氰基（electron withdrawing cyano group），该修饰策略有望降低该染料被单线态氧介导降解的敏感性。X射线晶体结构（X-ray crystal structure）分析显示，α-CN-TO显著偏离平面构象，而TO则以平面构象（planar conformation）结晶。α-CN-TO在粘性甘油-水溶剂（viscous glycerol/water solvent）中保留了母体染料TO的荧光生成特性（fluorogenic behavior），但直接光照与间接漂白实验（direct irradiation and indirect bleaching studies）表明，α-CN-TO对可见光与单线态氧基本呈惰性。此外，α-CN-TO的非平面构象减弱了其与DNA以及此前筛选获得的TO结合蛋白（TO-binding protein）的非特异性结合（nonspecific binding）与荧光激活作用，且在HeLa细胞培养体系（HeLa cell culture）中呈现较低的背景荧光（background fluorescence）。随后，研究人员利用α-CN-TO筛选得到一种可结合该染料并激活其荧光信号的全新蛋白质。相较于同类的TO/蛋白荧光模块（fluoromodule），该新型α-CN-TO/蛋白荧光模块展现出更优异的光稳定性。上述特性表明，α-CN-TO与特异性RNA、蛋白结合伴侣（binding partners）联合使用时，可作为一款实用的荧光生成型染料，适用于体外及细胞内相关应用。从更广的视角来看，促进非平面构象的结构特征，可为降低阳离子染料（cationic dyes）与核酸及其他生物分子的非特异性结合提供一种有效的策略。