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).
