Generalist versus Specialist Self-Replicators.

Darwinian evolution, including the selection of the fittest species under given environmental conditions, is a major milestone in the development of synthetic living systems. In this regard, generalist or specialist behavior (the ability to replicate in a broader or narrower, more specific food environment) are of importance. Here we demonstrate generalist and specialist behavior in dynamic combinatorial libraries composed of a peptide-based and an oligo(ethylene glycol) based building block. Three different sets of macrocyclic replicators could be distinguished based on their supramolecular organization: two prepared from a single building block as well as one prepared from an equimolar mixture of them. Peptide-containing hexamer replicators were found to be generalists, i. e. they could replicate in a broad range of food niches, whereas the octamer peptide-based replicator and hexameric ethyleneoxide-based replicator were proven to be specialists, i. e. they only replicate in very specific food niches that correspond to their composition. However, sequence specificity cannot be demonstrated for either of the generalist replicators. The generalist versus specialist nature of these replicators was linked to their supramolecular organization. Assembly modes that accommodate structurally different building blocks lead to generalist replicators, while assembly modes that are more restrictive yield specialist replicators.

Stochastic Emergence of Two Distinct Self-Replicators from a Dynamic Combinatorial Library.

Unraveling how chemistry can give rise to biology is one of the greatest challenges of contemporary science. Achieving life-like properties in chemical systems is therefore a popular topic of research. Synthetic chemical systems are usually deterministic: the outcome is determined by the experimental conditions. In contrast, many phenomena that occur in nature are not deterministic but caused by random fluctuations (stochastic). Here, we report on how, from a mixture of two synthetic molecules, two different self-replicators emerge in a stochastic fashion. Under the same experimental conditions, the two self-replicators are formed in various ratios over several repeats of the experiment. We show that this variation is caused by a stochastic nucleation process and that this stochasticity is more pronounced close to a phase boundary. While stochastic nucleation processes are common in crystal growth and chiral symmetry breaking, it is unprecedented for systems of synthetic self-replicators.

Two Sides of the Same Coin: Emergence of Foldamers and Self-Replicators from Dynamic Combinatorial Libraries.

The ability of molecules and systems to make copies of themselves and the ability of molecules to fold into stable, well-defined three-dimensional conformations are of considerable importance in the formation and persistence of life. The question of how, during the emergence of life, oligomerization reactions become selective and channel these reactions toward a small number of specific products remains largely unanswered. Herein, we demonstrate a fully synthetic chemical system where structurally complex foldamers and self-replicating assemblies emerge spontaneously and with high selectivity from pools of oligomers as a result of forming noncovalent interactions. Whether foldamers or replicators form depends on remarkably small differences in building block structures and composition and experimental conditions. We also observed the dynamic transformation of a foldamer into a replicator. These results show that the structural requirements/design criteria for building blocks that lead to foldamers are similar to those that lead to replicators. What determines whether folding or replication takes place is not necessarily the type of noncovalent interaction, but only whether they occur intra- or intermolecularly. This work brings together, for the first time, the fields of replicator and foldamer chemistry.

Self-Sorting in Dynamic Combinatorial Libraries Leads to the Co-Existence of Foldamers and Self-Replicators.

Nature segregates fundamental tasks such as information storage/transmission and catalysis between two different compound classes (e.g. polynucleotides for replication and folded polyamides for catalysis). This division of labor is likely a product of evolution, raising the question of how simpler systems in which replicators and folded macromolecules co-exist may emerge in the transition from chemistry to biology. In synthetic systems, achieving co-existence of replicators and foldamers in a single molecular network remains an unsolved problem. Previous work on dynamic molecular networks has given rise to either self-replicating fibers or well-defined foldamer structures (or completely un-sorted complex systems). We report a system in which two cross-reactive dithiol (nucleobase- and peptide-based) building blocks self-sort into a replicator fiber and foldamer that both emerge spontaneously and co-exist. The self-sorting behavior remains prevalent across different building block ratios as two phases of emergence occur: replicator growth followed by foldamer formation. This is attributed to the autocatalytic formation of the replicator fiber, followed by enrichment of the system in the remaining building block, which is subsequently incorporated into a foldamer.

Chemical Fueling Enables Molecular Complexification of Self-Replicators*.

Unravelling how the complexity of living systems can (have) emerge(d) from simple chemical reactions is one of the grand challenges in contemporary science. Evolving systems of self-replicating molecules may hold the key to this question. Here we show that, when a system of replicators is subjected to a regime where replication competes with replicator destruction, simple and fast replicators can give way to more complex and slower ones. The structurally more complex replicator was found to be functionally more proficient in the catalysis of a model reaction. These results show that chemical fueling can maintain systems of replicators out of equilibrium, populating more complex replicators that are otherwise not readily accessible. Such complexification represents an important requirement for achieving open-ended evolution as it should allow improved and ultimately also new functions to emerge.

Caught in the Act: Mechanistic Insight into Supramolecular Polymerization-Driven Self-Replication from Real-Time Visualization.

Self-assembly features prominently in fields ranging from materials science to biophysical chemistry. Assembly pathways, often passing through transient intermediates, can control the outcome of assembly processes. Yet, the mechanisms of self-assembly remain largely obscure due to a lack of experimental tools for probing these pathways at the molecular level. Here, the self-assembly of self-replicators into fibers is visualized in real-time by high-speed atomic force microscopy (HS-AFM). Fiber growth requires the conversion of precursor molecules into six-membered macrocycles, which constitute the fibers. HS-AFM experiments, supported by molecular dynamics simulations, revealed that aggregates of precursor molecules accumulate at the sides of the fibers, which then diffuse to the fiber ends where growth takes place. This mechanism of precursor reservoir formation, followed by one-dimensional diffusion, which guides the precursor molecules to the sites of growth, reduces the entropic penalty associated with colocalizing precursors and growth sites and constitutes a new mechanism for supramolecular polymerization.

Spontaneous Emergence of Self-Replicating Molecules Containing Nucleobases and Amino Acids.

The conditions that led to the formation of the first organisms and the ways that life originates from a lifeless chemical soup are poorly understood. The recent hypothesis of "RNA-peptide coevolution" suggests that the current close relationship between amino acids and nucleobases may well have extended to the origin of life. We now show how the interplay between these compound classes can give rise to new self-replicating molecules using a dynamic combinatorial approach. We report two strategies for the fabrication of chimeric amino acid/nucleobase self-replicating macrocycles capable of exponential growth. The first one relies on mixing nucleobase- and peptide-based building blocks, where the ligation of these two gives rise to highly specific chimeric ring structures. The second one starts from peptide nucleic acid (PNA) building blocks in which nucleobases are already linked to amino acids from the start. While previously reported nucleic acid-based self-replicating systems rely on presynthesis of (short) oligonucleotide sequences, self-replication in the present systems start from units containing only a single nucleobase. Self-replication is accompanied by self-assembly, spontaneously giving rise to an ordered one-dimensional arrangement of nucleobase nanostructures.

Automated device for continuous stirring while sampling in liquid chromatography systems.

Ultra-performance liquid chromatography is a common analysis tool, and stirring is common in many laboratory setups. Here we show a device which enables continuous stirring of samples whilst inside an ultra-performance liquid chromatography system. Utilizing standard magnetic stirring bars that fit standard vials, the device allows for the automation of experimental setups that require stirring. The device is designed such that it can replace the standard sample holder and fits in its place, while being battery operated. The use of three-dimensional (3D) printing and commercially available parts enables low-effort and low-cost device production, as well as easy modifications. Testing the device was performed by video analysis and by following the kinetics of a dynamic combinatorial library that is known to be exquisitely sensitive to agitation, as a result of involving a fiber growth-breakage mechanism. Design files and schematics are provided.