Periodic Reporting for period 4 - illumizymes (Illuminating aptamers and ribozymes for biomolecular tagging and fluorogen activation)
Okres sprawozdawczy: 2020-08-01 do 2022-06-30
Also, we characterized a large Stokes shift fluorogenic RNA aptamer named Chili that binds the ligands in the protonated phenol form and exploits excited state proton transfer pathways to enable a more than 350-fold enhanced fluorescence emission from the phenolate form of the bound chromophore. The ligands feature a cationic aromatic side chain for increased RNA affinity and reduced magnesium dependence. Our results suggest that Chili might be a versatile tool for future imaging applications.
Altogether, we managed to achieve many impressive results that already led to 19 publications in renowned, high-ranking journals such as Nature, Nature Chemical Biology, Nature Communications, Nature Structural & Molecular Biology, Angewandte Chemie (among them a VIP Paper and three Hot Papers), and The Journal of the American Chemical Society. Further manuscripts are currently in preparation. Some of our findings can be regarded as important landmarks for future investigations (see next paragraph). Furthermore, we presented many of our exciting discoveries to the scientific community at important conferences, in invited key lectures given by the PI, and in oral and poster presentations given by the co-workers involved in the project. The summer school on nucleic acid chemistry and synthetic biology in 2019, offering lectures from renowned experts on a wide variety of topics around the chemistry of nucleic acids, was an ideal platform for networking and the exchange of research ideas and experience related to our project. Overall, the funding that was granted made it possible to achieve many trend-setting results and led to increased visibility of our research group.
A new high-throughput approach was developed in the project, named DZ-seq. The innovative character of the new approach becomes apparent from the fact that DZ-seq is applicable for comprehensive studies on huge combinatorial libraries and can also be used for the analysis of very different types of nucleic acids. Since the new profiling approach makes it possible to identify deoxyribozymes that would have remained inaccessible using conventional methods, it will largely expand the portfolio of catalytic nucleic acids as powerful tools for biochemical research.
The fluorogenic Chili RNA aptamer attracted attention as an versatile and efficient RNA mimics of fluorescent proteins that specifically bind and activate conditional fluorophores such as analogs of the green and red fluorescent protein chromophore 4-hydroxybenzylidene imidazolone (HBI). Chili exclusively binds the protonated phenol form of the HBI derivatives and exploits excited state proton transfer pathways to enable a more than 350-fold enhanced fluorescence emission from the phenolate form of the HBI chromophore. The elucidation of the structural and mechanistic basis of fluorescence activation in the Chili aptamer ligand complex will pave the way for future engineering of fluorogenic moduls for sensors and imaging applications.