First, we developed a new set of AdoMet analogs for enzymatic transfer photo-caging groups. These include the ortho-nitrobenzylgroup as well as red-shifted derivatives. We could show that several methyltransferases are promiscous and accept the cosubstrate analogs and transfer photo-caging groups to nucleic acids. We modified the N7 position of the guanosine of the 5' cap of mRNA (GpppG) using the enzyme Ecm1. This modification strongly interfered with cap-binding proteins. Upon irradiation with light, we found that the ortho-nitrobenzyl group and its derivatives did not release the anticipated product. We discovered benzophenone functioned as an unusual photo-caging group if placed at the N7 position of guanosine. These major findings were published in Angewandte Chemie (L. Anhäuser, N. Klöcker, F. Muttach, F. Mäsing, P. Špaček, A. Studer and A. Rentmeister, Angewandte Chemie International Edition 2020, 59, 3161-3165).
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.
