Project Details
Projekt
New DNA polymerases for the direct detection of epigenetic marks in RNA
Applicant
Professor Dr. Andreas Marx
Subject Area
Biological and Biomimetic Chemistry
Term
from 2015 to 2022
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 277366853
During the last decades the view of nucleic acids as sole carrier of sequence information was inaccurate. In particular, RNA has been found to bear a considerably broader range of cellular functions, including the catalysis of biochemical reactions, the control of post-transcriptional modifications, and the regulation of gene expression. In fact, more than 150 enzymatically introduced and chemically distinct nucleotide modifications have been identified in cellular RNA. Modified nucleotides increase the structural versatility and chemical diversity of RNA molecules to allow for their multifarious cellular functions. In that way, they can fine-tune RNA folding as well as RNA-RNA and RNA-protein interactions. Moreover, highly dynamic nucleotide modifications in protein-coding RNAs are proposed to control mRNA metabolism and thereby regulate gene expression.A bottleneck along these lines is that available methods for the high-precision detection of these modifications are very laborious thereby hampering progress. The objective of this project is to develop widely applicable means that allow for the reliable detection of RNA modifications within RNA. In the first funding phase we were successful in “evolving” a reverse transcriptase enzyme that exhibits specific reverse transcription-signatures as a response to encountering an m6A-RNA modification. In close collaboration with the group of Mark Helm (University Mainz) and Yuri Motorin (L’Université de Lorraine, Nancy) within the SPP we could show that the engineered enzyme delivers prominent RT-signatures at m6A sites in different sequence contexts, while not exerting elevated error rates opposite unmodified nucleotides and the majority of the other modified nucleotides. This allows mapping of m6A sites by next generation sequencing.In the next funding phase we aim at a) the optimisation of the evolved 1st generation enzyme for enabling broad applications in the detection of m6A sites and b) Generation of a reverse transcriptase variant that is responsive to pseudouridine. Pseudouridinylation in mRNA has been suggested to perform important regulatory roles in mRNA metabolism, and thus, reliable techniques that allow detection of pseudouridine will spur the field. Thereby we will follow the experimental routes that were developed in the first funding phase including combinatorial protein design, screening and biochemical characterization. At the end of the project, we will be able to provide the community with enzymes that are tailored for the site selective detection of m6A and pseudouridine which will be beneficial for future applications. In addition, we will have gained more insight into the function of a DNA polymerase that is used in many biotechnological applications.
DFG Programme
Priority Programmes
