Periodic Reporting for period 4 - RNActivate (Optochemical control of cell fate by activation of mRNA translation)
Período documentado: 2022-12-01 hasta 2023-11-30
The goal of this project was to bring eukaryotic mRNA under the control of light to trigger efficient ectopic translation with spatio-temporal resolution in cells and in vivo. In addition to labeling and tracking subpopulations of cells, the potential of this technology to control and to manipulate cell fate by locally producing proteins responsible for cell death, genome engineering, and cell migration was explored. We used cultured cells and one-cell stage zebrafish embryos that can be easily injected with mRNA and activated translation by light with spatio-temporal precision. Our approach overcomes previous limitations of photo-inducible mRNA translation and enables researchers to manipulate mammalian cells or a transparent developing organism at the molecular level.
Furthermore, we could enzymatically photocage the N6-position of adenosine at internal positions in mRNA using METTL3-METTL14. In this case, irradiation with light led to the anticipated removal of the photo-caging group. The photocaging group impaired reverse transcription and this impairment was released after irradiation (A. Ovcharenko, F. P. Weissenboeck and A. Rentmeister, Angewandte Chemie International Edition 2021, 60, 4098-4103). The photocaging of the N6-position of adenosine also worked with DNA. Importantly, we developed a strategy to enzymatically make the AdoMet analogs with photocaging groups. To achieve this, we engineered methionine adenosyl transferases (MATs) from different organisms and solved the crystal structure. These results are the basis for metabolic labeling, because they allow us to now start from methionine analogs which are stable and cell-permeable, whereas the AdoMet analogs have limited stability and are not cell permeable. These results were published in Angewandte Chemie and featured in a press release and on the cover page (F. Michailidou, N. Klöcker, N. V. Cornelissen, R. K. Singh, A. Peters, A. Ovcharenko, D. Kümmel and A. Rentmeister, Angewandte Chemie International Edition 2021, 60, 480-485).
With respect to the 5' cap, we achieved enzymatic photocaging of the N2 position of m7G using the enzyme GlaTgs2-Var. This allowed us for the first time reconstitute the free 5' cap by irradiation. We could produce mRNAs with this photocaged 5' cap by transcriptional priming and observed that their translation was impaired. Upon irradiation with light in vitro, we observed an increase in translation of the mRNA. However, when this irradiation was performed on cells, the net effect on translation of photocaged mRNAs was negligible. We attributed this to the incomplete release of free 5' cap in combination with the negative effect of UV light on translation. The results were published in ChemBioChem (N. Klocker, L. Anhauser and A. Rentmeister, Chembiochem 2023, 24, e202200522).
Based on the insights gained from the above results, we realized that we needed to develop photocaged 5' caps that are more efficiently released, ideally by light that is not stressful to cells. We anticipated that connecting the photocleavable group by a self-immolative linker might improve the release of free 5' cap. A carbamate would present such a self-immolative linker, as it drives the reaction by CO2 release, however, it cannot be obtained by enzymatic conversion. We therefore shifted the project to a more chemical approach and achieved the complete chemical synthesis of FlashCaps. FlashCaps feature the 5' cap architecture with a photocleavable group at the N2 position connected by a carbamate. FlashCaps exhibited the desired properties: They prohibit binding to the translation initaion factor 4E and resist cleavage by decapping enzymes. They are compatible with the general and efficient production of mRNAs by in vitro transcription. In FlashCap-mRNAs, the single photocaging group abrogates translation in vitro and in mammalian cells without increasing immunogenicity. Irradiation restores the native cap, triggering efficient translation. FlashCaps overcome the problem of remaining sequence or structure changes in mRNA after irradiation that limited previous designs. These results were published in Nature Chemistry (N. Klocker, F. P. Weissenboeck, M. van Dulmen, P. Spacek, S. Huwel and A. Rentmeister, Nat Chem 2022, 14, 905-913.) They demonstrate that FlashCaps offer a route to regulate the expression of any given mRNA and to dose mRNA therapeutics with spatio-temporal control.
We achieved the enzymatic formation of AdoMet analogs with photocleavable groups by protein engineering of MAT. This will enable the intracellular transfer of photocleavalbe groups via metabolic labeling.
With FlashCaps, we developed a new and general approach to control translation of ectopic mRNA by light. FlashCaps now offer a route to regulate the expression of any given mRNA and to dose mRNA therapeutics with spatio-temporal control. They are compatible with all common procedures for making and handling mRNAs. We validated their functionality in different mammalian cell lines and zebrafish. Future work aims at using local activation of translation to manipulate cellular processes by light in living mammalian cells and transparent developing embryos.