Periodic Reporting for period 4 - EPiR (The Chemical Basis of RNA Epigenetics)
Reporting period: 2022-04-01 to 2023-11-30
Our overarching goal of the project is 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 want 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 is 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 is 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 will learn how the chemical structures such as DNA, RNA and the ribosome and hence life evolved and we will 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.
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. 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. Comm. 2019), 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 interest in the international press. The article was highlighted not only in a large number of scientific journals but also in the daily news. This publication formed next the basis to investigate the chemical synthesis of these modified bases as phosphoramidites and subsequently to explore their incorporation into RNA. Publications in this direction appeared in Angew. Chem. as a premium journal in organic chemistry and short, very competitive results were published in Chem. Comm. A further highlight of our study was the synthesis of the highly modified non-canonical nucleosides galactosyl- and mannosyl queuosine, which are sugar modified nucleobases present in the anticodon loop of tRNA. Here we could show that the chemical structure of mannosyl queuosine reported in the literature is wrong. We are in the process of correcting the chemical structure by total synthesis and direct comparison of the synthetic material with material isolated from various organs of higher organisms.
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. 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. Comm. 2019), 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 interest in the international press. The article was highlighted not only in a large number of scientific journals but also in the daily news. This publication formed next the basis to investigate the chemical synthesis of these modified bases as phosphoramidites and subsequently to explore their incorporation into RNA. Publications in this direction appeared in Angew. Chem. as a premium journal in organic chemistry and short, very competitive results were published in Chem. Comm. A further highlight of our study was the synthesis of the highly modified non-canonical nucleosides galactosyl- and mannosyl queuosine, which are sugar modified nucleobases present in the anticodon loop of tRNA. Here we could show that the chemical structure of mannosyl queuosine reported in the literature is wrong. We are in the process of correcting the chemical structure by total synthesis and direct comparison of the synthetic material with material isolated from various organs of higher organisms.
Our main goal in the second funding phase is now to further explore and develop the prebiotic routes to non-canonical nucleosides, specifically to the pyrimidines. We plan to reduce the chemistry to a simplicity that proves that these complex molecules can indeed form without human intervention, just driven by wet-dry cycles, hot-cold cycles and pH-shifts, which have all likely occurred on the prebiotic earth. We want to create the nucleobases that are forming RNA by just fluctuations of these parameters without the need of laboratory equipment or a chemist that controls and interferes with the individual reactions. Our goal is to fully simulate a prebiotic synthesis of the nucleobases using a flow reactor. We hope that these studies will influence the research field similar to the Urey-Miller experiment. Our goal is to create an “early earth simulator” that creates the complex nucleosides and nucleotides and potentially even small RNA strands. We want to shift prebiotic chemistry into a new dimension in order to show that primitive chemical conditions can provide rapidly the building blocks of the RNA world.
After having incorporated the modified nucleosides into RNA we are planning to move further into biology in order to investigate their function in more detail. Our goal is to study how the modified bases are interconverted in a living cell. We want to understand the reader, writer and eraser proteins that are binding to modified bases in order to elucidate the biological effect. The synthetic methods that were developed in the course of the 1st part of project have moved us far beyond the state of the art. They will be finally the basis for the synthesis of novel cyclic dinucleotide building blocks such as cGAMP derivatives, which may put us into the position to open up a new chapter in immuno-oncology. Our new synthetic methods are hoped to enable the efficient synthesis of cGAMP prodrug derivatives, which can enter cells to either stimulate or reduce the cellular immune-response against pathogens and cancer. A main result of our prebiotic and synthetic studies of modified bases may be the possibility to even synthesize immuno-stimulating cyclonucleotides that will help us to fight cancer.
After having incorporated the modified nucleosides into RNA we are planning to move further into biology in order to investigate their function in more detail. Our goal is to study how the modified bases are interconverted in a living cell. We want to understand the reader, writer and eraser proteins that are binding to modified bases in order to elucidate the biological effect. The synthetic methods that were developed in the course of the 1st part of project have moved us far beyond the state of the art. They will be finally the basis for the synthesis of novel cyclic dinucleotide building blocks such as cGAMP derivatives, which may put us into the position to open up a new chapter in immuno-oncology. Our new synthetic methods are hoped to enable the efficient synthesis of cGAMP prodrug derivatives, which can enter cells to either stimulate or reduce the cellular immune-response against pathogens and cancer. A main result of our prebiotic and synthetic studies of modified bases may be the possibility to even synthesize immuno-stimulating cyclonucleotides that will help us to fight cancer.