Periodic Reporting for period 4 - DynaGrow (Dynamic Growth and Replication in Coacervate Protocells)
Reporting period: 2024-09-01 to 2025-02-28
Replication and division are two of the most fundamental properties of living systems. Without replication, Darwinian evolution would not be possible, and life could never have reached the degree of complexity we see today. However, exactly how mixtures of non-living molecules developed the ability to replicate and divide, remains one of the biggest mysteries in modern science. Various molecular replicators have been investigated previously, but they are all destined to become extinct by dilution, since they lack a surrounding compartment that divides spontaneously during replication.
In this proposal, we aim at developing a new class of coacervate-based protocells that are capable of active growth and template-directed replication. The coacervates we propose here are condensed liquid droplets with a unique dual role: they act as a compartment that holds together and concentrates the template molecules and the building blocks, and they provide the right chemical environment for the replication reactions to take place at an appreciable rate.
The overall objective of this ERC proposal is to develop coacervate-based protocells that are capable of self-replication and evolution, as a physicochemical platform to study the link between compartmentalization and replication and the general principles underlying the emergence of living cells. The achieve this overall objective, we have 1) developed a wide range of chemically active coacervates that are capable of growth through chemical and enzymatic reactions, including reduction/oxidation, phosphorylation, native chemical ligation, and (de)acetylation. We have also 2) developed new methodologies to quantify the volume fraction of coacervates, the surface charge of coacervates and the binding strength of ions and small molecules to coacervate-forming molecules. Together with an in silico platform to model chemical reaction networks in coacervates, this enables us now to look at the rate of self-replication by RNA inside coacervates. Finally, 3) we developed two types of light-switchable coacervates to locally control birth and death rates in coacervate solutions to explore primitive evolution in coacervate populations.
In this proposal, we aim at developing a new class of coacervate-based protocells that are capable of active growth and template-directed replication. The coacervates we propose here are condensed liquid droplets with a unique dual role: they act as a compartment that holds together and concentrates the template molecules and the building blocks, and they provide the right chemical environment for the replication reactions to take place at an appreciable rate.
The overall objective of this ERC proposal is to develop coacervate-based protocells that are capable of self-replication and evolution, as a physicochemical platform to study the link between compartmentalization and replication and the general principles underlying the emergence of living cells. The achieve this overall objective, we have 1) developed a wide range of chemically active coacervates that are capable of growth through chemical and enzymatic reactions, including reduction/oxidation, phosphorylation, native chemical ligation, and (de)acetylation. We have also 2) developed new methodologies to quantify the volume fraction of coacervates, the surface charge of coacervates and the binding strength of ions and small molecules to coacervate-forming molecules. Together with an in silico platform to model chemical reaction networks in coacervates, this enables us now to look at the rate of self-replication by RNA inside coacervates. Finally, 3) we developed two types of light-switchable coacervates to locally control birth and death rates in coacervate solutions to explore primitive evolution in coacervate populations.
Several coacervate systems that show dynamic growth have been developed. This dynamic growth is manifested as a gradual increase in size of the coacervate protocells as a result of chemical reactions taking place either inside the protocells or in their immediate vicinity. The chemical reactions that can drive growth are diverse, ranging from reactions between small, prebiotically relevant molecules to enzyme-catalyzed conversions. Importantly, we discovered that the coacervates can function as novel types of catalysts: by concentrating substrates and providing a unique physicochemical environment for the reactions, they are able to accelerate chemical reactions by orders of magnitude. When these reactions give rise to new coacervate material, the coacervate protocells grow autocatalytically. Growth has been studied at single droplet level to afford a detailed understanding of the underlying mechanisms. The growth can be tuned by controlling the reaction rates, from minutes to days.
A versatile peptide-based synthon for simple coacervates with a stickers-and-spacer architecture was developed. By varying the amino acids in the stickers, various condensate shapes can be formed, including coacervates, star-shaped needles and amorphous aggregates. By including a disulfide bond in the spacer, exchange reactions are possible with potential for replication and evolution. Some simple coacervates could undergo a liquid-to-solid transformation, nucleated within the coacervates, and leading to well-defined fibers emerging from the droplets.
A versatile peptide-based synthon for simple coacervates with a stickers-and-spacer architecture was developed. By varying the amino acids in the stickers, various condensate shapes can be formed, including coacervates, star-shaped needles and amorphous aggregates. By including a disulfide bond in the spacer, exchange reactions are possible with potential for replication and evolution. Some simple coacervates could undergo a liquid-to-solid transformation, nucleated within the coacervates, and leading to well-defined fibers emerging from the droplets.
A short peptide derivative capable of forming simple coacervate compartments can be considered a breakthrough for the field of protocells and origin of life. The idea that coacervates could have served as protocellular compartments dates back nearly 100 years to Alexander Oparin. However, until now, all examples of coacervates were either formed with long synthetic polymers or complex and non-prebiotic molecules, such as PDDA, PLys, ATP and ELPs. Therefore, the coacervate hypothesis for the origin of life has long been considered unrealistic, because of the incompatibility with small molecules. We showed that short peptide derivatives with only 4 amino acids can undergo liquid-liquid phase separation and spontaneously form coacervate droplets under ambient conditions (pH 7, room temperature, weak salinity) that contain up to 75% water. The high water content enables uptake of active biomolecules and the coacervates function as catalytic microreactors for anabolic reactions. This puts the coacervate hypothesis for the origin of life back into a new perspective. Moreover, the enhanced reaction rates open the way for further development of coacervates as innovative green catalysts.
In addition, several new methods have been developed to quantify the volume fraction of coacervates, the surface charge of coacervates and the binding strength of ions and small molecules to coacervate-forming molecules. Together with an in silico platform to model chemical reaction networks in coacervates, this enables us now to look at the rate of self-replication by RNA inside coacervates. This opens the way for new rational design of coacervate-forming molecules and chemically active coacervates.
In addition, several new methods have been developed to quantify the volume fraction of coacervates, the surface charge of coacervates and the binding strength of ions and small molecules to coacervate-forming molecules. Together with an in silico platform to model chemical reaction networks in coacervates, this enables us now to look at the rate of self-replication by RNA inside coacervates. This opens the way for new rational design of coacervate-forming molecules and chemically active coacervates.