Final Report Summary - REPLI (Self replication in dynamic molecular networks) The discovery of the peptide-based replicator that emerges from a disulfide dynamic combinatorial library has opened many possibilities for research both into the mechanism of the system as well as the expansion of the system’s complexity. One of the major developments was proving that the studied replicators are indeed exponential. The extensive seeding experiments as well as simulations established our replicators as one of the very few that are truly exponential without the need of changing the conditions during the time of the reaction. Exponential replication is necessary to achieve evolution in a scenario where replicators compete with each other. In order to have competition we developed new replicators with different peptide sequences and different overall structure. Changing the peptide sequence allowed to create replicator molecules of different ring sizes that can catalyze their own formation as well as cross catalyze other replicators. These systems were extensively studied in isolation and also in systems with more than one building block. In systems where different building blocks are mixed together we observe formation of structural mutants that not only differ in macrocycle size but also in the building block content within the macrocycle, opening a new dimension in the structural diversity of the replicators. We pioneered flow systems where mixtures of different replicators are being fed the corresponding building blocks creating a scenario where competition for available resources dictates the outcome of the system. The basis for the self-replication of our systems lays in mechanosensitivity. In the first reported replicator the mechanical energy necessary for breaking the fibers and generating the fiber ends that promote replicator formation was delivered by stirring or shaking. Since then we have developed advanced Couette cell methodology that allows us to deliver mechanical energy in a controlled manner and greater efficiency. This approach allows us to generate fibers of controlled length with surprisingly narrow length distributions not found in other supramolecular 1-D structures. The Couette cell experiments give us insight into the effect of mechanical energy in the replication mechanism. We also showed that replication can be triggered through the addition of signal molecules. Two approaches have been developed. One in which the assembly of the replicator is promoted through the addition of a template molecule that amplifies the production of the replicator by binding to it. This raises the concentration of the replicator above its critical aggregation concentration. Once this concentration is exceeded the replicator can persist even in the absence of the molecule that triggered its emergence. In the second approach the signal molecule does not interact directly with the replicator molecules, but with the building blocks that interfere with replicator formation. In this case the signal molecule causes the amplification of a synthetic receptor that binds it, sequestering the building blocks from the mixture, leaving behind the building blocks needed by the replicator. Finally, we established the methodology for self-replication under far-from-equilibrium conditions through implementing replication-destruction regimes in two different ways. This methodology will be instrumental in making the next step in replicator chemistry: achieving Darwinian evolution.The results described above place our work at the forefront of replicator research and systems chemistry and bring us a step closer to the tantalizing goal of synthesizing life de-novo.