Development of cap-caged-mRNA for optochemical control of translation in eukaryotic cells

• Applicant: Professorin Dr. Andrea Rentmeister
• Subject Area: Biological and Biomimetic Chemistry ; Biochemistry
• Term: from 2017 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 390706730

• Final Report Year: 2023

Final Report Abstract

Light is a versatile regulatory element, because it is fully orthogonal to most cellular components, noninvasive, controllable in timing and localization to tissues, cells and even subcellular regions. Various optochemical approaches have allowed the control of gene expression at the level of DNA, mRNA, and protein with spatio-temporal control. However, an efficient method to induce translation of exogenous mRNA with spatio-temporal control was missing. We developed an innovative approach for triggering translation of exogenous mRNA with light. Towards this end, we first developed a chemo-enzymatic route to modify the 5′ cap with photocleavable groups and block its interaction with cap-binding proteins, like the translation initiation factor eIF4E. Consequently, the translation of mRNAs with these modified 5′ caps was silenced. As proposed, we used the highly promiscuous N7 cap methyltransferase Ecm1 and synthesized novel analogs of the cosubstrate S-adenosyl-L-methionine (AdoMet). In parallel, we established enzymatic transfer of photocleavable groups to the N2 position of the m7G of the 5′ cap, using the methyltransferase variant GlaTgs-V34A. When we established light-triggered removal of photoresponsive groups to reconstitute the canonical 5′ cap, we found that ortho-nitrobenzyl (ONB)-derivatives were not suitable for photodeprotection of N7G, but caused decomposition of the purine ring upon prolonged irradiation. Instead, we discovered that the benzophenone-group and a coumarine-derivative were removable by light from N7G. For all photocaged 5′ caps tested, we could demonstrate co-transcriptional incorporation into mRNA. We generated reporter mRNAs with differently modified 5′ caps and transfected mammalian cells to evaluate the translational output with or without irradiation. In all cases, the translation of cap-caged mRNAs was strongly reduced without irradiation. For N2-ONB-cap-modified mRNA, we could show proof-of-concept that light-mediated (partial) removal of the photo-caging group slightly increases translation of the respective mRNA in mammalian cells. Most importantly, N7-coumarine-cap-modified mRNA was fully translationally silenced. By irradiation of cells transfected with this cap-caged mRNA, the translation was efficiently turned on, as shown by an increase in the amount of reporter protein detected in irradiated cells. Finally, by combining ONB-derived and coumarine-based photo-cleavable groups we could demonstrate wavelength-selective activation of translation of two different reportermRNAs in cells.

Publications

• A Benzophenone‐Based Photocaging Strategy for the N7 Position of Guanosine. Angewandte Chemie International Edition, 59(8), 3161-3165.
  Anhäuser, Lea; Klöcker, Nils; Muttach, Fabian; Mäsing, Florian; Špaček, Petr; Studer, Armido & Rentmeister, Andrea
  
• Combining Chemical Synthesis and Enzymatic Methylation to Access Short RNAs with Various 5′ Caps. ChemBioChem, 20(13), 1693-1700.
  Muthmann, Nils; Guez, Théo; Vasseur, Jean‐Jacques; Jaffrey, Samie R.; Debart, Françoise & Rentmeister, Andrea
  
• Multiple covalent fluorescence labeling of eukaryotic mRNA at the poly(A) tail enhances translation and can be performed in living cells. Nucleic Acids Research, 47(7), e42-e42.
  Anhäuser, Lea; Hüwel, Sabine; Zobel, Thomas & Rentmeister, Andrea
  
• Engineered SAM Synthetases for Enzymatic Generation of AdoMet Analogs with Photocaging Groups and Reversible DNA Modification in Cascade Reactions. Angewandte Chemie International Edition, 60(1), 480-485.
  Michailidou, Freideriki; Klöcker, Nils; Cornelissen, Nicolas V.; Singh, Rohit K.; Peters, Aileen; Ovcharenko, Anna; Kümmel, Daniel & Rentmeister, Andrea
  
• Tag‐Free Internal RNA Labeling and Photocaging Based on mRNA Methyltransferases. Angewandte Chemie International Edition, 60(8), 4098-4103.
  Ovcharenko, Anna; Weissenboeck, Florian P. & Rentmeister, Andrea
  
• Chemo‐Enzymatic Modification of the 5′ Cap Maintains Translation and Increases Immunogenic Properties of mRNA. Angewandte Chemie International Edition, 60(24), 13280-13286.
  van Dülmen, Melissa; Muthmann, Nils & Rentmeister, Andrea
  
• CAPturAM, a Chemo‐Enzymatic Strategy for Selective Enrichment and Detection of Physiological CAPAM‐Targets. Angewandte Chemie International Edition, 62(4).
  Muthmann, Nils; Albers, Marvin & Rentmeister, Andrea
  
• Enzymatic Modification of the 5’ Cap with Photocleavable ONB‐Derivatives Using GlaTgs V34A. ChemBioChem, 24(2).
  Klöcker, Nils; Anhäuser, Lea & Rentmeister, Andrea
  
• Light‐Activated Translation of Different mRNAs in Cells via Wavelength‐Dependent Photouncaging. Angewandte Chemie International Edition, 62(5).
  Bollu, Amarnath; Klöcker, Nils; Špaček, Petr; P., Weissenboeck Florian; Hüwel, Sabine & Rentmeister, Andrea
  
• Quantification of mRNA cap-modifications by means of LC-QqQ-MS. Methods, 203, 196-206.
  Muthmann, Nils; Špaček, Petr; Reichert, Dennis; van Dülmen, Melissa & Rentmeister, Andrea