Final Report Summary - OPTORIBO (Optogenetic control of cellular behaviour by allosteric ribonucleic acid assemblies)
In Optogenetics, light-sensitive channel proteins are used to control the electrophysiology of cells, especially neurons, and, thus, to analyze neuronal function in a spatiotemporal manner. Likewise, soluble photoreceptor proteins, e.g. those having so-called light, oxygen, voltage (LOV) domains are used to generate fusion proteins enabling light-control of enzymes in cells. However, to date, optogenetic solutions that address endogenous, intracellular biomolecules especially RNA molecules in a universal fashion remain elusive. Although approx. 75% of all transcripts in mammalian cells are not translated into proteins, but harbor biochemical functions tools to investigate these in a spatiotemporal and minimal invasive manner are elusive. In this regard, the OptoRibo project generated and characterized a series of RNA aptamers by a so-called systematic evolution of ligands by exponential enrichment (SELEX) process that interact with the bacterial photoreceptor protein PAL in a light-dependent manner. PAL has a unique protein architecture, with a C-terminal LOV and a N-terminal PAS domain flanking a central ANTAR domain, which is thought to mediate RNA aptamer binding. The PAL protein was identified in the gram-positive actinobacterium Nakamurella multipartitia. For gaining light-control of RNA function in eukaryotes, we validated that PAL can be expressed in mammalian cells and maintain reversible light sensitivity. The identified RNA aptamers were found to share two different RNA motifs, which most likely fold into short hairpin structures and the corresponding minimal motifs are built from 17 and 19 nucleotides, respectively. Due to these small sizes and compact structures, the hairpin motifs were implemented into other cellular RNA molecules to gain light control over RNA function. To this end, we embedded the RNA aptamers into the 5’-untranslated region of a reporter gene’s mRNA to enable light-control of protein expression in mammalian cells and demonstrated that the PAL-RNA platform can be employed to regulate gene expression at the RNA level as a function of light in mammalian cells. These results conjoin RNA biology with optogenetic regulation, thereby paving the way towards hitherto inaccessible optoribogenetic modalities. This approach is not limited to achieving regulation on the level of mRNA but will be broadly applicable to many more RNA molecules, for example micro RNA and lncRNA to gain spatiotemporal control of RNA function inside cells.