Periodic Reporting for period 2 - ActiDrops (Synthetic Active Droplets Inspired by Life) Reporting period: 2021-08-01 to 2023-01-31 Summary of the context and overall objectives of the project Active droplets are made of molecular building blocks that are coupled to a metabolic chemical reaction network (Image 1). This means that non-active precursors are converted into droplet molecules at the expense of energy from a fuel. Importantly, the droplet molecule formed is not stable and spontaneously reacts back to the non-active precursor. Overall, the droplet molecule can only exist as long as the fuel is present. In biology, membrane-less organelles like P-granules or Cajal bodies rely on active droplet formation to compartmentalize their processes since they do not contain lipid membranes. Despite the crucial role of active droplets in cell biology, the precise mechanism regulating their behavior remains elusive. Some theoretical models have already predicted that active droplets all evolve to the same optimal size, or even more interestingly, they can spontaneously self-divide when energy is abundant. All of these exciting properties are essential to the functioning of life. Therefore, if we could translate these behaviors into synthetic materials, we would obtain a better understanding of the mechanism underlying active self-assembly and its role in biology and the origin of life.The goal is thus to develop synthetic active droplets and study the “life-like” behaviors that emerge from their active nature. These include the spontaneous emergence when fuel is added, their “death” when fuel is scarce, their collective behavior, their spontaneous self-division when energy is abundant, their active growth at the expense of others, and their ability to converge to one optimal size (Image 1). To be successful, the following four specific objectives are being tackled: 1. To tune the residence time of the active droplet material by developing a library of active modules with varying residence times. 2. To synthesize active droplets based on the active modules previously mentioned. Droplets that emerge in response to fuel and disappear in the absence of fuel are expected at this point. 3. To understand the mechanism of accelerated ripening of active droplets and to model how membrane-less organelles can rapidly grow and how the number and location of these organelles can be regulated. 4. To understand how activation and deactivation kinetics can control the size of the active droplets, and whether active droplets can spontaneously self-divide. The successful completion of the project will have an impact far beyond supramolecular chemistry. ActiDrop will develop new strategies to create and control materials with life-like properties for the material science community. For biophysics, the active droplets could serve as a model for membrane-less organelles. For the community working on the origin of life question, ActiDrops may unravel mechanisms by which active droplets can spontaneously self-divide without the need for cell machinery. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far ActiDrop uses a simple and versatile fuel-driven chemical reaction cycle (CRC) developed in Prof. Boekhoven Lab. The chemical reaction cycle comprises the activation of a carboxylate molecule at the expense of one molecule of fuel and simultaneously, the deactivation of the anhydride product by its spontaneous hydrolysis. The loss of anionic charges upon activation of the precursor drives the self-assembly process. Over the last months, ActiDrops focused mainly on developing a library of active modules with different residence times that could combine with the fuel-driven CRC previously mentioned and the synthesis of active droplets based on these active modules. Moreover, some progress has also been made to understand the mechanism of accelerated ripening of active droplets.ActiDrops can already use a library of active modules with vastly different residence times. We can work with active modules that survive seconds and others that stand for hours. All of them are compatible with the CRC. We already combined them with different building blocks and successfully synthesized different types of active droplets. - Firstly, we mainly used long aliphatic building blocks to develop some active oil droplets that were phase-separated from water. We confirmed that both the activation and deactivation occur outside the active droplets since the droplets do not contain water. Then, we moved forward with active oil droplets and proved the accelerating ripening that we had already hypothesized. The active droplets we created grow exceptionally quickly, meaning orders of magnitude faster than expected for Ostwald ripening. More interestingly, the rapid growth of the droplets can be regulated by the residence time of the active module. Moreover, we proposed a mechanism for the active ripening for a single batch of fuel and when continuous fuel was applied (ChemSystemsChem 2021). In addition, using this type of active oil droplets, we also observed two unexpected phenomena that we identified as “parasitic” and “self-emulative” behaviors (ChemicalScience 2021).- Secondly, we synthesized active complex coacervates by combining a cationic peptide product created upon fuel addition with an anionic polymer (e.g. RNA). In contrast to active oil droplets, the emerging coacervate droplets comprise up to 95% water. That means that the anhydride product can be hydrolyzed inside the droplet. Hence, the activation takes part outside the droplet, whereas deactivation takes place within the droplet. ActiDrops’ active complex coacervates form spontaneously and decay in the absence of fuel. Excitingly, some other active behaviors, reminiscent of life-like behaviors, were observed like fusion or vacuole formation. Our fuel-driven complex coacervates can also transiently up concentrate functional RNA (NatCommun 2020). 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) Despite the recent developments in active materials, examples of synthetic active droplets did not exist, but ActiDrops made them possible. We successfully synthesized for the first time active oil droplets and active complex coacervate droplets. The vastly different water content of these two types of droplets endows them with very different properties. Concerning active oil droplets, ActiDrops has already confirmed the mechanism of accelerated ripening after a systematic study with varying the residence times of the active modules. As hypothesized, the accelerating ripening effect increases with decreasing residence times. Currently, the project focuses on conducting similar experiments in a restricted volume. We expect these experiments to be more biologically relevant to that of a cell. In the coming months, we aim to test the role of accelerated ripening as a mechanism by which cells control their membrane-less organelles. Regarding active complex coacervates, ActiDrops did manage to synthesize the first example of active complex coacervates and observed some life-like reminiscent behaviors. We envisioned these active coacervate droplets to be an excellent model for membrane-less organelles. We reported as well some preliminary design rules for chemically fueled complex coacervates (JACS 2021). These rules will make it easier to obtain such coacervates and, therefore, expand their use and potential applications.ActiDrops currently studies how the CRC, i.e. how the residence time influences the complex coacervate behavior. ActiDrops aims to test for size control of active coacervates in the coming months by regulating the residence time and ultimately test for spontaneous self-division. Schematic representation of active droplets and their life-like properties
