Periodic Reporting for period 5 - EPiR (The Chemical Basis of RNA Epigenetics)
Berichtszeitraum: 2023-12-01 bis 2024-09-30
Our overarching goal of the project was to create a comprehensive picture of how non-canonical DNA and RNA bases, which are today central elements of all genetic systems, have originated and to elucidate their biological function. These non-canonical nucleosides form a second layer of (epi)genetic information that is largely unexplored. In the first objective, we wanted to understand the biological epigenetic function of these unusual structures in the genetic system in order to learn how they influence the contemporary transcriptional and translational systems. A second objective was to understand how these non-canonical nucleosides were integrated into the chemical structures that establish the genetic system in the first place. Did they evolve late in order to help life to cope with increasing complexity or were they already formed on the early earth as companion and competitor molecules to the canonical Watson-Crick bases? Were they essential for allowing life to form in the way that we know today? The central goal of the project was to develop a comprehensive picture of how the chemical complexity of the genetic system evolved and how it today encodes genetic information. This included the development of new concepts of how the ribosome evolved in which the genetic information is translated into a peptide sequence with the essential help of the non-canonical nucleobases. We wanted to learn how the chemical structures such as DNA, RNA and the ribosome and hence life evolved and we aimed to get new insights into the deeper function of the epigenetic chemical components of the genetic system. This will lead to the discovery of new targets and pathways to treat today untreatable diseases. Today, we can proudly say that we achieved astonishing milestones towards these goals. We established entirely new concepts that have not been thought of before. We did not close the chapters we have opened. Instead, we established new research fields and novel technology that will thrive in the future.
We started our research program with a closer look at the potential prebiotic origin of the canonical and non-canonical nucleosides. The first idea was to decipher a prebiotically plausible chemical pathway from very primitive molecules that were likely present on the early earth, to complex nucleosides and modified nucleosides including amino acid modified nucleosides. This involved the development of completely new early earth compatible synthetic pathways to nucleosides. These needed to be water-compatible (what classical synthetic routes to nucleosides are not!) and which are able to proceed without human intervention. We then continued to analyse the function of the non-canonical nucleosides that we find embedded in DNA and RNA in modern time. This forced us to develop new chemical synthesis pathways to the non-canonical nucleosides to give phosphoramidite derivatives that were needed to incorporate the non-canonical nucleosides into synthetic DNA and RNA strands. With these strands in hand, we started to investigate how these modified bases influence the physical properties of RNA. We wanted to learn whether these modified bases are able to form stable base pairs or if they induce alternative oligonucleotide structures, which are important for their function. With chemical synthesis of RNA containing modified bases available, we already started to perform biological studies in order to elucidate the function of these non-canonical bases during transcription and translation.
We indeed achieved development of a new prebiotic route to pyrimidine and purine nucleosides, based on simple molecules like formic acid, urea, hydroxylurea, sodium nitrite and isocyanate (Science 2016/2019). This allowed us to generate purine and pyrimidine bases under prebiotically plausible conditions. We could show that this chemistry generates not only the canonical uridine bases A, G, C and U but also a large number of modified nucleobases (Nat.Commun. 2018), which are those that are indeed found today in contemporary RNA. We could therefore formulate the theory that the modified RNA bases that are today found in human RNA are indeed likely relics of an early earth chemistry and that they must have been present as competitor and companion molecules of the canonical bases. The corresponding publication in Science 2019 has received tremendous attention in the international press. The article was highlighted not only in a large number of scientific journals but also in the daily news.
Our next milestone achievement was based on our ability to incorporate highly modified nucleosides into polymers, on which we developed a prebiotically plausible scenario of an RNA-peptide world and the transmission of chirality. Those high impact publications have earned us the participation in a television documentary ("Life from Outer Space", ARTE) , highlighting important aspects of our work. We were also invited to organize the Leopoldina conference on the "Origin of Life". A further highlight of our research was the synthesis of the highly modified non-canonical nucleosides glutamyl-, galactosyl- and mannosyl queuosine, which are hyper-modified nucleobases present in the anticodon loop of tRNA. Here we could show by total synthesis and direct comparison of the synthetic material with material isolated from various organs that the chemical structure of mannosyl queuosine reported in the literature was wrong. Our expertise in nucleic acids synthesis and analysis allowed us to successfully enter the world of immunology, to which we contributed novel STING agonists based on the second messenger cGAMP and the elucidation of the role of RNase T2 in innate immunity.
We indeed achieved development of a new prebiotic route to pyrimidine and purine nucleosides, based on simple molecules like formic acid, urea, hydroxylurea, sodium nitrite and isocyanate (Science 2016/2019). This allowed us to generate purine and pyrimidine bases under prebiotically plausible conditions. We could show that this chemistry generates not only the canonical uridine bases A, G, C and U but also a large number of modified nucleobases (Nat.Commun. 2018), which are those that are indeed found today in contemporary RNA. We could therefore formulate the theory that the modified RNA bases that are today found in human RNA are indeed likely relics of an early earth chemistry and that they must have been present as competitor and companion molecules of the canonical bases. The corresponding publication in Science 2019 has received tremendous attention in the international press. The article was highlighted not only in a large number of scientific journals but also in the daily news.
Our next milestone achievement was based on our ability to incorporate highly modified nucleosides into polymers, on which we developed a prebiotically plausible scenario of an RNA-peptide world and the transmission of chirality. Those high impact publications have earned us the participation in a television documentary ("Life from Outer Space", ARTE) , highlighting important aspects of our work. We were also invited to organize the Leopoldina conference on the "Origin of Life". A further highlight of our research was the synthesis of the highly modified non-canonical nucleosides glutamyl-, galactosyl- and mannosyl queuosine, which are hyper-modified nucleobases present in the anticodon loop of tRNA. Here we could show by total synthesis and direct comparison of the synthetic material with material isolated from various organs that the chemical structure of mannosyl queuosine reported in the literature was wrong. Our expertise in nucleic acids synthesis and analysis allowed us to successfully enter the world of immunology, to which we contributed novel STING agonists based on the second messenger cGAMP and the elucidation of the role of RNase T2 in innate immunity.
Funding through the ERC has allowed us to develop entirely novel concepts about the emergence of the building blocks for the origin of life on early earth. Our results led to a completely new chemistry and directed us into the realms of the origin of translation, where we postulated a new concept of a co-developing RNA-peptide world. We were able to provide new tools for nucleic acids quantification by mass spectrometry that is used by many researchers in the field and generated novel modalities for the immunological treatment of cancer. All those developments did not come to an end with the end of the funding period. Instead, they fuelled totally novel research directions. Just to give a few examples, we have started investigating our prebiotic chemistry under the more realistic assumption that the atmosphere on early earth was not highly reducing, which in our opinion was a common misconception in the field. We further developed novel tools for nucleic acids delivery, e.g. of siRNA to treat SARS-CoV-2. We study the compartmentation of nucleic acids in the context of the formation of the first living cells. Finally, we developed new robust and gentle sequencing technology for epigenetic information. The improvement of a novel epigenetic drug has just been awarded with a PoC grant by the European Union. Our efforts culminated recently in establishment of a center for nucleic acid therapeutics (CNATM), funded by the German Ministry for Education and Research. With all required modesty, we believe that our work had an important impact on the field of nucleic acid chemistry and therapeutics and has strengthened the European chemical biology community.