GFP-like fluorescent proteins and their molecular mimics have revolutionized bioimaging research, but their emissions are largely limited in the visible to far-red region, hampering the in vivo applications in intact animals. Herein, we structurally modulate GFP-like chromophores using a donor–acceptor–acceptor (D–A–A′) molecular configuration to discover a set of novel fluorogenic derivatives with infrared-shifted spectra. These chromophores can be fluorescently elicited by their specific interaction with G-quadruplex (G4), a unique noncanonical nucleic acid secondary structure, via inhibition of the chromophores’ twisted-intramolecular charge transfer. This feature allows us to create, for the first time, FP mimics with tunable emission in the near-infrared (NIR) region (Em_max = 664–705 nm), namely, infrared G-quadruplex mimics of FPs (igMFP). Compared with their FP counterparts, igMFPs exhibit remarkably higher quantum yields, larger Stokes shift, and better photostability. In a proof-of-concept application using pathogen-related G4s as the target, we exploited igMFPs to directly visualize native hepatitis C virus (HCV) RNA genome in living cells via their in situ formation by the chromophore-bound viral G4 structure in the HCV core gene. Furthermore, igMFPs are capable of high contrast HCV RNA imaging in living mice bearing a HCV RNA-presenting mini-organ, providing the first application of FP mimics in whole-animal imaging.

中文翻译：

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荧光蛋白的近红外核酸模拟物的开发，用于具有开启荧光的病毒 RNA 的体内成像

类 GFP 荧光蛋白及其分子模拟物已经彻底改变了生物成像研究，但它们的发射在很大程度上仅限于可见光至远红色区域，阻碍了在完整动物体内的应用。在这里，我们使用供体-受体-受体（D-A-A'）分子构型在结构上调节 GFP 样发色团，以发现一组具有红外位移光谱的新型荧光衍生物。这些生色团可以通过它们与 G-四链体 (G4) 的特异性相互作用而被荧光激发，G-quadruplex (G4) 是一种独特的非规范核酸二级结构，通过抑制生色团的扭曲分子内电荷转移。这一特性使我们第一次能够在近红外 (NIR) 区域 (Em _{max ) 中创建具有可调发射的 FP 模拟}= 664–705 nm），即 FP 的红外 G-四链体模拟物（igMFP）。与 FP 对应物相比，igMFP 表现出显着更高的量子产率、更大的斯托克斯位移和更好的光稳定性。在使用病原体相关 G4 作为目标的概念验证应用程序中，我们利用 igMFP 直接可视化活细胞中的天然丙型肝炎病毒 (HCV) RNA 基因组，通过它们在原位形成的发色团结合的病毒 G4 结构在活细胞中形成。丙肝病毒核心基因。此外，igMFP 能够在带有 HCV RNA 呈递微型器官的活体小鼠中进行高对比度 HCV RNA 成像，这是 FP 模拟物在整个动物成像中的首次应用。