Chemo-enzymatic synthesis of base-modified NTPs for the post-transcriptional 5′-cap and 3′-tail modification of mRNA

• Applicant: Dr. Nicolas Vincent Cornelissen
• Subject Area: Biological and Biomimetic Chemistry
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 572249202

Following the first approved mRNA-based vaccines against SARS-CoV-2, numerous clinical trials for mRNA therapeutics are ongoing for the treatment of infectious diseases, cancers and cardiovascular diseases. Molecular innovation is needed to enhance the stability of mRNAs and to increase the protein production in cells. This will boost therapeutic mRNAs and enable protein replacement therapies in the future. In this research project, we develop chemo-enzymatic strategies for the synthesis of base-modified GTP and ATP analogues (WP1), that can be used as novel mRNA building blocks. The combination of nucleoside monophosphates synthesis with a polyphosphate kinase catalysed 5′-phosphorylation enables simple, fast and inexpensive access to base-modified NTP analogues. While previous research often used UTP or CTP derivatives to reduce immunogenicity in the entire mRNA sequence, we want to target two hallmarks of eukaryotic mRNA, namely the mRNA 5′-cap and the 3′-poly(A) tail. This strategy has the advantage that the mRNA can be modified post-transcriptionally and that the modified NTPs do not need to be accepted by the polymerase (e.g. T7, SP6) used for the in vitro transcription (IVT). After transfection into cells, the modified part of the mRNA is not decoded by the ribosome, circumventing a second current limitation in the use of modified nucleotides. The GTP analogues are applied for the post-transcriptional 5′-capping of mRNAs (WP2) aiming at increasing the stability against decapping enzymes (e.g. Dcp2) and increasing the affinity for the eukaryotic translation initiation factor (eIF4E). The ATP analogues are applied for the generation of mRNAs with hypermodified poly(A) tails (WP3), to increase the stability against the CCR4-NOT complex, while maintaining the interactions needed for efficient translation. Both the head and tail modifications of mRNA aim to increase protein production of the modified mRNAs and will be tested in mammalian cell lines.

• DFG Programme: Research Grants