Self-cleaving Molecular Beacons for amplified nucleic acid detection

由DNA/RNA模板控制的化学反应为核酸诊断、编码药物库的开发以及潜在的治疗方法的应用奠定了基础。在本项目中，我们将开发一种用于超高灵敏度检测特定核分子的光化学扩增系统。长期目标之一是检测细胞中低丰度的RNA分子。为此，我们将构建分子信标探针（molecular beacon probes, scMB），使其与靶模板杂交后发生裂解反应。该方法只需要一个反应性寡核苷酸探针，这与现有的DNA/ rna模板反应需要2到4个探针进行扩增形成了对比。自裂解反应的产物对模板的亲和力将首次低于反应前的探针。在不受产物抑制影响的情况下，模板将发挥催化活性。裂解选项的引入将为广泛使用的单分子分子信标探针提供更高的灵敏度。自裂分子信标（scMB）含有一个易受光催化裂解反应影响的连接基团SL。荧光团F一方面作为报告基团，另一方面作为光触发电子转移反应的引发剂。在没有目标的情况下，scMB采用发夹结构，该结构以允许荧光团激发态快速耗尽的方式定位猝灭基团。scMB与靶杂交后打开，猝灭剂与F分离，荧光发生。在靶结合状态下，F可以光催化SL的裂解。裂解产物被新的scMB探针取代，提供来自F的信号放大。在本研究项目中，我们将优化光裂解连接基团，鉴定光催化能力强的荧光团，并开发不同的方法来合成scMB探针。通过改变scMB探针的结构，我们将揭示RNA模板诱导的scMB切割反应对高效率的要求。最终，我们将使用scMB探针检测细胞中的mRNA分子。

Chemical reactions that are controlled by DNA/RNA templates lay the foundations for applications in nucleic acid diagnostics, the development of encoded drug libraries and, potentially, for theranostics approaches. In this project, we will develop a photochemical amplification system for the ultra high sensitivity detection of specific nucleic molecules. One of the long-term objectives is to detect low abundance RNA molecules in cells. For this purpose, we will construct molecular beacon probes (scMB), which undergo a cleavage reaction upon hybridization with the target = template. The method requires only a single reactive oligonucleotide probe, which stands in contrast to existing DNA/RNA-templated reaction requiring two to four probes for amplification. The products of the self-cleavage reaction will have, for the first time, lower affinity for the template than the probe before reaction. Without being affected by product inhibition, the template will exert catalytic activity. The introduction of a cleavage option will provide the widely used unimolecular molecular beacon probes with enhanced sensitivity.A self-cleaving molecular beacon (scMB) contains a linker group SL that is susceptible to a photocatalytic cleavage reaction. A fluorophore F serves, on the one hand, as reporter group and, on the other hand, as initiator of a phototriggered electron transfer reaction. In the absence of target, the scMB adopts a hairpin structure, which positions a quencher group in a way that allows rapid depletion of the fluorophore’s excited state. The scMB opens upon hybridization with target, the quencher separates from F and fluorescence can occur. In the target-bound state F can photocatalyze the cleavage of SL. The cleavage products are displaced by new incoming scMB probes providing for amplification of signals from F. In this research project we will optimize photocleavable linker groups, identify photocatalytically competent fluorophores and develop divergent methods for the synthesis of scMB probes. By varying the structure of scMB probes we will unravel the requirements for high efficiency in RNA template-induced scMB cleavage reactions. Ultimately, we will use the scMB probes for detection of mRNA molecules in cells .