Metabolic labeling of RNA by means of synthetic and bioorthogonally reactive nucleoside as fluorogenic probes in living cells

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In order to investigate biological processes of nucleic acids, in particular RNA, in their natural environment, inside living cells, they have to be conjugated with fluorescent labels. The metabolic labeling has been established for RNA. However, in nearly all published examples the metabolic labeling of RNA and DNA was –independently of the applied reaction type- performed with fixed cells, and not in living cells. In this project, the metabolic labeling of RNA will be further developed by means of synthetic and bioorthogonally reactive nucleosides to a fluorogenic tool. By combination with FISH probes, RNA transcript will be not only visualized as snapshots in fixed cells but also in living cells. The metabolically labeled RNA works as fluorogenic energy donor, the FISH probes as energy acceptor. The labeling applies Diels-Alder reactions with inverse electron demand and “photoclick” reactions, because they were successfully approved for metabolic labeling in living cells, and thus are the organic-chemical basis for this project. Cyclopropenes will be used as bioorthogonally reactive groups, because they are only slightly bigger than vinyl groups, they are both sufficiently reactive and sufficiently stable functional groups, they can be synthesized in a straightforward way, and thus, they are suitable for metabolic labeling. 1-Methylcyclopropenes will be used for metabolic Diels-Alder reactions and 3-methylcyclopropenes for metabolic “photoclick” reactions. In vitro experiments characterize their reactivity and kinetics. The metabolic labeling will be analyzed in HeLa cells by RNA hydrolysis. The fluorogenic properties is evaluated in combination with FISH probes on selected mRNA transcripts and miRNA sequences.