Posttranscriptional Control of Gene Function With Light-dependent RNA Editing and Reversal of Psoralen Crosslinks

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Our major objective is the development of methods for controlling posttranscriptional gene regulation with light. Recently, we have established directed A-to-I-RNA editing as a tool for transcript repair and RNA reprogramming. With this proposal we aim to photocontrol RNA editing (first in vitro, later in cell culture) by incorporating photolabile protection groups on the essential benzylguanine residue and on the phosphordiester backbone. If successful this will enable the assembly of enzymatically functional guideRNA-deaminase conjugates inside the cell under spatial and temporal control with light. This would pave the way towards numerous attractive applications even beyond RNA editing.Furthermore we have established Psoralen-crosslinking for the photocontrol of DNA function during the last two years. We now aim to transfer the technology to the RNA-level. For this we will generate a small crosslinked hairpin motif for its later introduction into larger RNA constructs. This may allow us to control the translation of such constructs with light. The method has the potential to complement classical methods that rely on photolabile protection groups and may thus offer access to novel applications. In contrast to classical photocaging, already a single crosslink can freeze a nucleic acid reliably and completely in an energetically unfavored secondary and tertiary structure.