Development of new fluorescent probes for nucleic acids based on aminophthalimide as isosteric base substitution and photostable cyanine-styryl dyes

荧光标记不仅在化学生物分析和分子诊断中发挥着重要作用，而且在实际蓬勃发展的活细胞分子成像研究领域也发挥着重要作用。该项目的中心目标仍然是开发新的核酸荧光探针，这种探针基于其优化的结构和光学性质，适用于化学生物学，并可以取代较弱的市售荧光探针。该项目在第一个资助期的重点是开发新的DNA荧光探针，该探针基于与嘌呤等同的氨苯酰亚胺，以及作为DNA合成后修饰的光稳定的花青素-苯乙烯染料，如项目报告所述，并在出版物中有记录。在第二个资助期内，我们建议将重点转移到RNA上，并开发这些RNA荧光探针。具体的挑战仍然分为两个子项目：(A)具有荧光团的等构探针，其DNA/RNA碱基的大小大约较小，但仍然显示出大的斯托克斯位移和适用的量子产率很重要，因为它们只略微改变核酸的结构及其生物相互作用（子项目A）。在第一个资助期，我们建立了氨基眼酰亚胺作为DNA的碱基替代物。现在应该开发并建立RNA探针。(B)对于生物正交和“可点击”荧光探针，不仅光稳定性对单分子光谱长期细胞成像的应用很重要，而且荧光特性也决定了这些应用。后者将是第二个筹资期的新标准。荧光探针有一个很大的优势，那就是只有那些与RNA（和DNA）结合的荧光团被增强的发射所照亮（子项目B）。总而言之，该项目将为RNA生物分析和成像提供新的强大的荧光探针。

Fluorescent labeling plays not only a major role in chemical bioanalytics and molecular diagnostics, but also in the actually strongly expanding research field of molecular imaging of living cells. The central goal of this project remains to be the development of new fluorescent probes for nucleic acids, which are suitable for chemical biology based on their optimized structure and optical properties, and can replace weaker, but commercially available fluorescent probes. The focus of this project in the first funding period was the development of new fluorescent probes for DNA based on aminophthalimide that is isosteric to purines, and photostable cyanine-styryl dyes as postsynthetic modifications for DNA, as described in the project report and documented by publications. During the second funding period we propose to shift the focus onto RNA and develop these fluorescent probes for RNA. The particular challenges are still divided into two subprojects: (A) Isosteric probes with fluorophores that have approximately the small size of DNA/RNA bases but nonetheless show large Stokes’ shifts and applicable quantum yields are important because they do only slightly alter the structure of nucleic acids and their biological interactions (subproject A). In the first funding period, we established aminophthalimide as base surrogate for DNA. It should now be developed and established as RNA probe. (B) For the bioorthogonally and “clickable” fluorescent probes not only photostability is important for the application in long-lasting cellular imaging by single molecule spectroscopy, but also fluorogenic properties are determing these applications. The latter will be a new criterium for the second funding period. Fluorogenic probes have the great advantage that only those fluorophores are lighting up by their enhanced emission which are conjugated to RNA (and DNA) (subproject B). Taken together, this project will deliver new and powerful fluorescent probes for RNA bioanalytics and imaging.