Self-cleaving Molecular Beacons for amplified nucleic acid detection

• Applicant: Professor Dr. Oliver Seitz
• Subject Area: Biological and Biomimetic Chemistry
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 456693735

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 .

• DFG Programme: Research Grants