Novel peptide replicators from dynamic combinatorial libraries

This thesis focuses on the emergence of novel peptide self-replicators from dynamic combinatorial libraries. Thiol functionalised peptide building blocks, first reported by our group in 2010, form the basis of all chapters. The work performed here has two main objectives: First, to deepen the insights obtainable from self-replicating systems and second, to use peptide building blocks to mimic life-like features such as replication. Alongside this, we developed novel strategies to access novel replicators and to dictate their properties

Chemical Cascading Between Polymersomal Nanoreactor Populations

[EN] Harnessing interactions of functional nano-compartments to generate larger particle assemblies allows studying diverse biological behaviors based on their population states and can lead to the development of smart materials. Herein, thiol-functionalized polymersome nanoreactors are utilized as responsive organelle-like nano-compartments-with inherent capacity to associate into larger aggregates in response to change in the redox state of their environment-to study the kinetics of cascade reactions and explore functions of their collective under different population states. Two nanoreactor populations, glucose oxidase- and horseradish peroxidase-loaded polymersomes, are prepared, and the results of their cascading upon addition of glucose are investigated. The kinetics of resorufin production in associated polymersomes and non-associated polymersome populations are compared, observing a decreased rate upon association. For the associated populations, faster chemical cascading is found when the two types of nanoreactors are associated in a concerted step, as compared to sequential association. The addition of competing agents such as catalase impacts the communication between non-associated polymersomes, whereas such an effect is less pronounced for the associated ones. Altogether, the results showcase the impact of collective associations on enzymatic cascading between organelle-like nanoreactors.Y.A. and A.L.-L. contributed equally to this work. The authors would like to acknowledge the support from the Dutch Ministry of Education, Culture, and Science (Gravitation program 024.001.035 and Spinoza premium) and the ERC Advanced Grant (Artisym 694120).A.L.-L. acknowledges support from the MSCA Cofund project oLife, which has received funding from the European Union's Horizon 2020 research and innovation program under the Grant Agreement 847675; and the Maria Zambrano Program from the Spanish Government funded by NextGenerationEU from the European Union. Dr. Imke Pijpers is thanked for cryo-TEM imaging. Dr. Pascal Welzen is acknowledged for advice and useful discussion on polymer and polymersome preparation.Altay, Y.; Llopis-Lorente, A.; Abdelmohsen, LKEA.; Van Hest, JC. (2023). Chemical Cascading Between Polymersomal Nanoreactor Populations. Macromolecular Chemistry and Physics. 224(1):1-5. https://doi.org/10.1002/macp.20220026915224

An Optical Probe for Real-Time Monitoring of Self-Replicator Emergence and Distinguishing between Replicators

[Image: see text] Self-replicating systems play an important role in research on the synthesis and origin of life. Monitoring of these systems has mostly relied on techniques such as NMR or chromatography, which are limited in throughput and demanding when monitoring replication in real time. To circumvent these problems, we now developed a pattern-generating fluorescent molecular probe (an ID-probe) capable of discriminating replicators of different chemical composition and monitoring the process of replicator formation in real time, giving distinct signatures for starting materials, intermediates, and final products. Optical monitoring of replicators dramatically reduces the analysis time and sample quantities compared to most currently used methods and opens the door for future high-throughput experimentation in protocell environments

Structural and Spectroscopic Properties of Assemblies of Self-Replicating Peptide Macrocycles

Self-replication at the molecular level is often seen as essential to the early origins of life. Recently a mechanism of self-replication has been discovered in which replicator self-assembly drives the process. We have studied one of the examples of such self-assembling self-replicating molecules to a high level of structural detail using a combination of computational and spectroscopic techniques. Molecular Dynamics simulations of self-assembled stacks of peptide-derived replicators provide insights into the structural characteristics of the system and serve as the basis for semiempirical calculations of the UV-vis, circular dichroism (CD) and infrared (IR) absorption spectra that reflect the chiral organization and peptide secondary structure of the stacks. Two proposed structural models are tested by comparing calculated spectra to experimental data from electron microscopy, CD and IR spectroscopy, resulting in a better insight into the specific supramolecular interactions that lead to self-replication. Specifically, we find a cooperative self-assembly process in which β-sheet formation leads to well-organized structures, while also the aromatic core of the macrocycles plays an important role in the stability of the resulting fibers

Parasitic Behavior of Self-Replicating Molecules

Self-replication plays a central role in the origin of life and in strategies to synthesize life de novo. Studies on self-replication have focused mostly on isolated systems, while the dynamics of systems containing multiple replicators have received comparatively little attention. Yet most evolutionary scenarios involve the interplay between different replicators. Here we report the emergence of parasitic behavior in a system containing self-replicators derived from two subtly different building blocks 1 and 2. Replicators from 2 form readily through cross-catalysis by pre-existing replicators made from 1. Once formed, the new replicators consume the original replicators to which they owe their existence. These results resemble parasitic and predatory behavior that is normally associated with living systems and show how such lifelike behavior has its roots in relatively simple systems of self-replicating molecules

Existing Self-Replicators Can Direct the Emergence of New Ones

The study of the interplay between different self-replicating molecules constitutes an important new phase in the synthesis of life and in unravelling the origin of life. Here we show how existing replicators can direct the nature of a newly formed replicator. Starting from the same building block, 6-ring replicators formed when the mixture was exposed to pre-existing 6-membered replicators, while pre-formed 8-membered replicators funneled the building block into 8-ring replicators. Not only ring size, but also the mode of assembly of the rings into stacks was inherited from the pre-existing replicators. These results show that the nature of self-replicating molecules can be strongly influenced by the interplay between different self-replicators, overriding preferences innate to the structure of the building block

Emergence of a New Self-Replicator from a Dynamic Combinatorial Library Requires a Specific Pre-Existing Replicator

Our knowledge regarding the early steps in the formation of evolvable life and what constitutes the minimal molecular basis of life remains far from complete. The recent emergence of systems chemistry reinvigorated the investigation of systems of self-replicating molecules to address these questions. Most of these studies focus on single replicators and the effects of replicators on the emergence of other replicators remains under-investigated. Here we show the cross-catalyzed emergence of a novel self-replicator from a dynamic combinatorial library made from a threonine containing peptide building block, which, by itself, only forms trimers and tetramers that do not replicate. Upon seeding of this library with different replicators of different macrocycle size (hexamers and octamers), we observed the emergence of hexamer replicator consisting of six units of the threonine peptide only when it is seeded with an octamer replicator containing eight units of a serine building block. These results reveal for the first time how a new replicator can emerge in a process that relies critically on the assistance by another replicator through cross-catalysis and that replicator composition is history dependent

Adaptive polymeric assemblies for applications in biomimicry and nanomedicine

Dynamic and adaptive self-assembly systems are able to sense an external or internal (energy or matter) input and respond via chemical or physical property changes. Nanomaterials that show such transient behavior have received increasing interest in the field of nanomedicine due to improved spatiotemporal control of the nanocarrier function. In this regard, much can be learned from the field of systems chemistry and bottom-up synthetic biology, in which complex and intelligent networks of nanomaterials are designed that show transient behavior and function to advance our understanding of the complexity of living systems. In this Perspective, we highlight the recent advancements in adaptive nanomaterials used for nanomedicine and trends in transient responsive self-assembly systems to envisage how these fields can be integrated for the formation of next-generation adaptive stimuli-responsive nanocarriers in nanomedicine

Chemical Cascading Between Polymersomal Nanoreactor Populations

Harnessing interactions of functional nano-compartments to generate larger particle assemblies allows studying diverse biological behaviors based on their population states and can lead to the development of smart materials. Herein, thiol-functionalized polymersome nanoreactors are utilized as responsive organelle-like nano-compartments—with inherent capacity to associate into larger aggregates in response to change in the redox state of their environment—to study the kinetics of cascade reactions and explore functions of their collective under different population states. Two nanoreactor populations, glucose oxidase- and horseradish peroxidase-loaded polymersomes, are prepared, and the results of their cascading upon addition of glucose are investigated. The kinetics of resorufin production in associated polymersomes and non-associated polymersome populations are compared, observing a decreased rate upon association. For the associated populations, faster chemical cascading is found when the two types of nanoreactors are associated in a concerted step, as compared to sequential association. The addition of competing agents such as catalase impacts the communication between non-associated polymersomes, whereas such an effect is less pronounced for the associated ones. Altogether, the results showcase the impact of collective associations on enzymatic cascading between organelle-like nanoreactors