Periodic Reporting for period 3 - ABIOS (ABIOtic Synthesis of RNA: an investigation on how life started before biology existed) Reporting period: 2021-02-01 to 2022-07-31 Summary of the context and overall objectives of the project The emergence of life is one of the most fascinating and yet largely unsolved questions in the natural sciences, and thus a significant challenge for scientists from many disciplines. There is growing evidence that ribonucleic acid (RNA) polymers, which are capable of genetic information storage and self-catalysis, were involved in the early forms of life. But despite recent progress, RNA synthesis without biological machineries is very challenging. The current project aims at understanding how to synthesize RNA in abiotic conditions. We will address problems associated with three critical aspects of RNA formation that I will rationalize at a molecular level: (i) accumulation of precursors, (ii) formation of a chemical bond between RNA monomers, and (iii) tolerance for alternative backbone sugars or linkages. These questions range from the formation of chemical bonds up to the stability of large biopolymers, and we thus need a multi-scale approach combining techniques that range from quantum calculations to large-scale all-atom simulations, employed together with efficient enhanced-sampling algorithms, forcefield improvement, cutting-edge analysis methods and model development. The objectives are the following: 1 • To explain why the poorly-understood thermally-driven process of thermophoresis can contribute to the accumulation of dilute precursors. 2 • To understand why linking RNA monomers with phosphoester bonds is so difficult, to understand the molecular mechanism of possible catalysts and to suggest key improvements. 3 • To rationalize the molecular basis for RNA tolerance for alternative backbone sugars or linkages that have probably been incorporated in abiotic conditions. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far We were able to initiate the three research axes, and we already made considerable progress. 1 • Thermophoresis: we established a robust strategy to reproduce a thermophoretic setting in atomistic molecular dynamics simulations, and we now routinely apply it to a large variety of small neutral solutes in aqueous solutions. We are currently accumulating data to assess the dependence of the thermophoretic behavior of a given molecule upon several factors: its size, its affinity for water, its mass, or its concentration. We aim to rationalize and to be able to predict the behavior of a given molecule against a temperature gradient. Our data so far, although not final, seem to suggest that existing models and theories are not necessarily valid for molecular solutes in water. 2 • Phosphoester bond formation: we started to tackle the very challenging issue of phosphoester bond reactivity. In order to be able to sample the reaction over relatively long timescale, we are currently relying on a strategy that consists in a semi-empirical description of atomic interactions. We spent a significant amount of time designing adequate reaction coordinates and finding an appropriate method to sample the reaction. This set-up was applied to a simple model reaction between a small alcohol molecule and phosphate. We were able to identify two different reaction pathways and mechanisms, that we now aim to characterize using more accurate quantum descriptions. The effect of pH or of activating leaving groups is currently under investigation. 3 • The final part aims to characterize the effect of chemical alterations in the sugar backbone of RNA. Before considering these modifications per se, we first focused on one of the most important aspects of RNA strands in abiotic conditions, which is the stability of duplex strands that occur in the final stage in the RNA duplication mechanism in abiotic conditions. Separation of these strands is very challenging without enzymes and requires high temperatures. In order to understand how chemical modifications would later influence the strand separation upon thermal excitation, we first wanted to develop a robust strategy to assess RNA duplexes separation as temperature increases. We adapted an original method that we had applied in the past to proteins, with extremely promising results that were obtained on small model strands of varying sequence. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) 1 • We have developed a unique strategy to address thermophoresis in all-atom molecular dynamics simulations. We aim to provide a new molecular model for thermophoresis in aqueous solutions. We then aim to target the more complicated case of charged molecules, for which electrophoretic forces also contribute to the observed concentration gradients. As a final stage of the project, we will thus be able to address molecular solutes in salt solutions, mimicking abiotic conditions. 2 • We plan to have a thorough understanding of phosophoester bond formation for the model reaction between phosphate and a small alcohol molecule, from the semi-empirical current description we have to more sophisticated and accurate description. A very promising aspect, that is currently under investigation, is the opportunity to develop reactive classical forcefields based on deep learning approaches.3 • Now that we have a robust set-up to study the thermal separation of RNA duplexes, we will modify existing RNA forcefields to account for chemical alterations of the sugar backbone. We should then be able to fulfill the goals of this part, i.e. to characterize the effect of these backbone modifications on the strand behavior.