Periodic Reporting for period 1 - DSR (Dissipative Self-Replication) Reporting period: 2017-04-01 to 2019-03-31 Summary of the context and overall objectives of the project The transition from chemistry into biology is one of the grand challenges in contemporary science. Approaching such a complex question would be easier if we had a clear definition of life. It is increasingly clear that we have to shed light on the missing link between the simplest living system and inanimate matter. In other words, we have to define all the essential ingredients that are necessary to fabricate life-like systems. The main prerequisites for life as we know it, are replication, mutation and selection. The systems should be able to undergo Darwinian evolution. Laboratory processes are mostly designed such that the (closed) system goes thermodynamically downhill. Yet the chemistry of life operates in a very different way: Most molecules from which living systems are constituted are turned over continuously and are not necessarily thermodynamically stable. The overall aim of this project was to achieve Darwinian evolution of self-replicating molecules. Thus, replication will be operated far-from-equilibrium under conditions where replication and replicator destruction occur simultaneously. In order to survive, replicators need to replicate faster than they are being destroyed. By allowing replicators to mutate and by exposing the mutant distributions to changing conditions, those mutants that replicate fastest in the new environment will thrive. Thus, the systems exhibit all aspects of Darwinian evolution: replication, mutation and selection. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far "The overall objective of the project was to achieve Darwinian evolution in a fully synthetic chemical system. In order to demonstrate this, we made use of a variety of different building blocks, that are equipped with two thiol groups. In order to operate the system under non-equilibrium conditions, an experimental CSTR setup was successfully constructed. The setup consisted of a stirring plate where the libraries are constantly agitated and two syringe pumps: the one is for infusion of the “food”, the other one is for withdrawal of a portion of the entire solution (D1.1). We performed flow experiments by mixing building blocks 1 and 2 at different flow rates. At lower flow rates, we observed that at the end of almost 2 turnovers, the replicator distribution was sustained. Upon increasing the flow rate, the replicators could be successfully sustained at the end of 3 turnovers, demonstrating that a steady state of replicators could be achieved away from thermodynamic equilibrium (M1.1). In parallel, the synthesis of building blocks 5 and 6 was performed and their self-assembly in mixed systems revealed an unexpected emergent behavior, the coexistence of two replicators. This was observed for a first time with disulfide based replicators. Experiments under flow conditions, by constant addition of momomers, suggested that replicator formation and destruction can be achieved under far-from-equilibrium conditions with a self-sorting system (M1.1).Subsequently, we aimed to create distributions of replicator mutants under non-equilibrium conditions. To achieve this, we have interplayed with the conditions such that a stationary state could be achieved that contains replicator mutants as well as ""food"". Through an interplay with the concentration of the building blocks, we sought to achieve a steady state. We were able to detect only 1-rich replicators at the final composition, showing that specific mutants can be sustained away from thermodynamic equilibrium (M2.1). An impact on the replicator composition has also been observed in the self-sorting system developed in work package 1. Under flow conditions, the tetramer replicator could be sustained in high concentrations, while the trimer remained at low concentrations during the flow (M2.1). This steady state can be altered by switching off the flow, resulting in the composition that the library exhibited before supplying any “food”. In order to achieve selection of the autocatalytic species, a collaboration was built with the research group of Professor Dieter Braun in Ludwig-Maximilians-Universität (LMU), Munich. Successful fabrication of thermal traps, compatible with disulfide chemistry was achieved (D3.1) resulting in accumulation of larger species (dimer). Regarding the more complex system involving the replicator assemblies, upon flowing in building blocks and flowing out shorter replicator fibers, we have observed significant accumulation at the bottom of the trap as a result of the thermal gradient generation, suggesting selective retention (M.3.1). We also focused on changing the environmental conditions in order to favor mutants over others. As evidenced using UPLC/MS, in the presence of 1.5 M guanidinium chloride (a strong denaturant on protein folding), a trimer replicator could be emerged, instead of an hexamer. Furthermore, in flow conditions, constant addition of guanidinium chloride could give rise to interplay with both autocatalytic species. Similar effects have been observed for the serine containing replicator, by using co-solvents (M.3.2).Chemically fueled self-replication has been approached through chemical degradation by reduction, accompanied with simultaneous oxidation. In order to demonstrate chemical fueling, we made use of the system developed in the work package 3, involving the formation of two self-replicators arising from building block 1 in the presence of high concentration of guanidinium chloride.The oxidant and reductant reagents were infused concurrently by separate syringe pumps at the same rate in order to maintain a steady oxidation state. A steady state could be maintained for several hours after adding the same equivalents of oxidant and reductant. It is worth mentioning that upon stopping the supply of fuel, the system relaxed back to the original replicator composition, dominated by the trimer replicator. These results establish the first example of chemically fueled dissipative self-replication in complex molecular networks (M4.1)." 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) The DSR represents the first approach to achieve Darwinian evolution in a system of fully synthetic self-replicating molecules. The results arising from this project represent an important step towards the creation of de novo life, where far-from-equilibrium self-replication is completely unprecedented. Until the end of the project, we expect to be able to couple the thermal traps developed in LMU in the research group of Professor Braun with on line UPLC measurements to monitor in situ changes in replicator composition upon redox gradient. Overall, we envision DSR to be a pioneering research program in the field of origin of life and systems chemistry and may be extended towards many other directions, such as metabolism (replicators could ‘”learn” how to catalyze chemical reactions that produce their own food) and prediction of evolutionary scenarios (Exploring multi-parameter space computationally). The general public is mostly familiar with the principles of Darwinian evolution, but very much intrigued by the question of the origin of life and all the important aspects surrounding this fundamental question. This was furthermore highlighted by the Dutch Research National Agenda (NWA), where the question regarding the origin of life is on top priority, after a survey taken on Dutch citizens . Thus, the results arised from this research will find immediate interest to a general audience and in the same time will raise the profile of (systems) chemistry in society.