Deciphering the 5ꞌ cap epitranscriptome by nanopore sequencing via chemo-enzymatic modification (DeCapNanoChem)

• Applicants: Professor Dr. Christoph Dieterich; Professorin Dr. Andrea Rentmeister
• Subject Area: Biochemistry
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 556609629

Modifications play crucial roles in the complex life of any RNA molecule. In particular, the landscape of mRNA modifications is more complex than initially thought. There is increasing evidence that this is also true for the 5′ cap together with the transcription start nucleotide, which can vary considerably in their methylation pattern. We therefore need to better understand the role of the 5′ cap methylation status for the life of individual transcripts. The novel 3rd generation sequencing technologies such as ONT Nanopore can directly read RNA, DNA or cDNA. Current challenges remain in sequencing and data analysis of 5′ ends of mRNAs on 3rd generation sequencing technologies. To the best of our knowledge, there is no full-length mRNA sequencing approach available that would capture the type of mRNA 5′ cap in detail. In this project, we propose to use the chemo-enzymatic modification of the 5′ cap to covalently append an entire oligonucleotide. The key idea is to sufficiently extend the 5′ end such that it can be accurately measured on the nanopore before the motor protein falls off. The project requires the development of chemical biology and bioinformatics methodology to capture, sequence and analyze the 5′ ends of single mRNA molecules. Importantly, by making the original 5′ end amenable to nanopore sequencing, we aim to distinguish between different types of 5′ caps by single molecule, direct sequencing.

• DFG Programme: Research Grants