Self-Replication out-of-Equilibrium
Yang, Shuo
DOI:
10.33612/diss.171627402
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2021
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1.1 Life and evolution
In the last several decades, even though many theories and experiments have been proposed1, the fascinating mystery of the origin of life is still far from resolved. The most difficult challenge is to seek powerful chemical tools as platform for the verification of the huge number of insightful theories, as experimental progress is much less developed than theoretical work. To develop such chemical systems, it is essential to capture the key elements of life, which is also the best we can do at this stage due to the inherent unimaginable complexity of living systems2. There is a general consensus among most scientists that replication, compartmentalization and metabolism are three features that are of key importance3. Replication is the ability to carry information and transfer it to the offspring. Compartmentalization is what allows separating living entities from the environment. The ability of consuming energy and staying away from thermodynamic equilibrium, is called metabolism. Progress has been made in constructing chemical systems that combine at most two of the three elements mentioned above, and so far metabolism has received the least attention. The next big stride is to develop a system integrating all three elements which would be a crucial step in bridging chemistry and biology.
In NASA’s famous definition, life is “a self-sustaining chemical system capable of Darwinian evolution”, in which the concept of Darwinian evolution is highlighted4. The core idea of Darwinian evolution is that processes of replication, mutation and selection lead to complexification and diversification of living systems. So far the most stunning examples of Darwinian evolution at the molecular level are the cross-replicators by Joyce5and cooperating replicators by Lehman6, both of which are based on RNA. To create purely chemical synthetic systems capable of undergoing Darwinian evolution remains a challenge: there are not too many efficient replicators that can be utilized, which makes it even more difficult to generate mutants, let alone bringing about selection in a formation-destruction regime in an out-of-equilibrium steady state. More details about Darwinian evolution in chemical systems are discussed insection 1.3.
1.2 Supramolecular systems chemistry
For the last centuries, research in chemistry has mostly focused on synthesis and investigation of specific molecules. The striking development of this traditional covalent chemistry has paved the way for the appearance of supramolecular chemistry, in which the interactions between molecules are the key point. As the complexity increases, approaches are required to study the way emergent properties may arise from mixtures of molecules. These approached
culminated in a new field called systems chemistry, a term that was first coined by von Kiedrowski7. The core of this new field is to bottom-up engineer complex fully synthetic systems with life-like characteristics, as life is the most fascinating feature that can emerge from complex chemical networks. Many classic supramolecular systems (molecular machines, self-assembly, etc) have been complemented with some of the most important features of life, such as replication (autocatalyis and nonlinear kinetics), compartmentalization (growth and division) and metabolism (dissipative chemistry). And now the trend is to integrate these subsystems to reach higher levels of complexity, where interesting, not necessarily predictable properties (even de-novo life) may emerge. Next, examples of subsystem coupling are described.
1.2.1 Molecular motion
As one of the most sophisticated systems in biology, molecular machines are appealing not only to biologist, but also to chemists. Many synthetic molecular machines have been developed, including walkers, pumps, transporters and muscles8. Importantly, the motion of biological molecular machines is usually chemically fueled, by chemical gradients or the hydrolysis of adenosine triphosphate (ATP). Recently Leigh reported how the unidirectional motion of a fully synthetic molecular motor can be driven by chemical reagents (Figure 1.1A)9. The system is composed of a small molecular ring threading onto a larger ring (as track). The track has two active sites (stations) at which the small ring can dock. There are molecular groups that act as signals at the stations which can be switched between ‘go’ and ‘stop’ using the fuel reagent Fmoc-Cl. When the ring is docked at the first station, the fueled reaction cannot progress due to the ring’s proximity, which keeps the sign stay in ‘go’ position and the ring can move to the second station. After docking at the second station, at the first station turns the signal to ‘stop’, which prevents the ring from going backward. Then as the signal at the second station changes to ‘go’, the ring will move further along the track, completing the cycle. The key point is to make sure that the forward motion is much faster than the reverse. The movement continues as long as the fuel Fmoc-Cl is supplied.
Another challenge of synthetic molecular machines is to amplify their motion to achieve effects at the macroscale. Giuseppone reported a gel that can reversibly expand and contract by integrating a unidirectional motor and a switchable modulator, with light control (Figure 1.1B)10. The gel is composed of a Feringa-type motor, a dithienylethene-based modulator and polyethyleneglycol chains. UV irradiation activates the motor and deactivates the modulator at the same time, resulting in the contraction of the polymer networks in a braiding fashion.
Irradiating the gel with visible light induces the ring-opening reaction of the modulator, which unbraids the polymer chains to the extended state. Macroscopic contraction and expansion cycles can be repeated under alternating irradiation of visible and UV light.
Figure 1.1 (A) A small molecular motor fueled by chemical input. Reproduced from ref 9. (B) Illustrations of light-driven
molecular motors responsible of the macroscopic contraction of a gel. Reproduced from ref 10.
1.2.2 Complex compartmentalised networks
Compartmentalisation, replication and metabolism are the three most important characteristics of life. Research on synthetic systems still mostly focused on either compartmentalisation or replication. However, to approach the challenging problems of the origin of life, it is necessary to merge two (or even three) of the features listed above. Next, examples of combinations of compartmentalisation with genetic systems or dissipative conditions are described.
Szostak demonstrated a homeostatic state of ribozyme-containing protocells (Figure 1.2A)11. Despite the volume change of the synthetic cells during growth and division, the activity of the entrapped ribozymes remained constant. The protocells are composed of fatty acid, glycerol ester and phospholipid. Inside the protocells there are ribozymes and a high concentration of short oligonucleotides, which upon duplex formation, deactivate the ribozymes. When the protocells are growing by fusing with empty vesicles, the dissociation of the duplex caused by dilution increases the ribozymes’ activity. They suggested that such mechanism featuring short oligonucleotides could have preceded modern RNA regulatory mechanisms. And it may make contribution to the development of nucleic acid-based protocells with increasing complexity.
Prins reported a self-assembly of vesicular reactors under dissipative conditions (Figure 1.2B)12. Vesicles are formed through the supramolecular interactions between adenosine triphosphate (ATP) and the surfactants. Potato apyrase can convert ATP to adenosine monophosphate (AMP) by enzymatic hydrolysis. The ATP acts as a chemical fuel without which the vesicles will disassemble. The results show that controlling the hydrolysis rate of ATP can regulate the lifetime of the vesicles. Specifically, there is an equilibrium between the ATP in the solution and the assembled ATP on the vesicles. Potato apyrase preferentially hydrolyzes ATP in the solution and when it is largely hydrolyzed due to concentration re-equilibration the assembled ATP will dissociates from the vesicles, resulting in the degradation of the vesicles. In addition, the vesicles can sustain a chemical reaction only when the system is under dissipative conditions provided by ATP as chemical fuel. Based on dissipative compartmentalisation, the implementation of chemical function is an important step towards more complex life-like synthetic systems.
Figure 1.2 (A) Illustrations of a protocellular system with homeostatic behaviour: the ribozyme activity stay constant
during volume changes. Reproduced form ref 11.(B) The transient formation of vesicular reactors mediated by ATP
hydrolysis. Reproduced form ref 12.
1.2.3 Oscillating systems
Oscillatory dynamics is a key feature of life and plays critical roles in cellular processes through controlling the functions of biomolecules in space and time. Over the past decades, research has focused on the ‘bottom-up’ approach to construct oscillating systems. The build up of a chemical oscillator requires coupling of positive feedback (autocatalysis) and negative feedback (inhibition). The early studies on oscillations were mostly using inorganic reactions, such as the Belousov-Zhabotinsky (BZ) reaction13. Recently, new oscillating systems have
been established using biomolecules or small organic molecules rather than inorganic building blocks, which are more similar to biological complex networks.
Huck reported a trypsin-based enzymatic oscillator and provided a modular approach to rationally design tunable chemical networks under flow conditions (Figure 1.3A)14. The positive feedback is the autocatalytic reaction of trypsin from inactive trypsinogen. For an oscillator it is essential to incorporate a negative feedback (inhibit trypsin) that is delayed with respect to the production of trypsin. To be able to tune the inhibition rate, the authors splitted the negative feedback into two steps: firstly, the lysine group on the proinhibitor was cleaved by trypsin, then the intermediate inhibitor was cleaved by a second enzyme aminopeptidase, resulting in an active inhibitor. The system showed sustained and rich oscillating behaviour by modulating the flow rates of the components.
In contrast to the above example in which biomolecules were used, Whitesides constructed an oscillator using small organic molecules exclusively (Figure 1.3B)15. The network shares the same principles of autocatalysis-inhibition as previous examples. An autocatalytic reaction generates amides and thiols from dialkyl disulfides and thioester. A trigger reaction (formation by a thiol from hydrolysis of a thiolester) control the autocatalytic growth rate. In the inhibition process the autocatalytic thiol is deactivated by conjugate addition to acrylamide. Besides oscillation of the concentration of thiol, the system can show bistability by tuning the rate of the inhibition reaction. Based on the nature of the molecules the authors argue that their system may have some prebiotic relevance.
Figure 1.3 Schematic representation of oscillating systems constructed by (A) biomolecules or (B) small organic
1.2.4 Reaction and diffusion
In living systems, reaction-diffusion represents an energy consumption process which leads to ordered patterns or structures. The reaction-diffusion process is intimately related to biological phenomena in living cells such as chemotaxis, cell division, signalling cascades and self-organization. Translating these notions into synthetic systems remains a challenge. Epstein has demonstrated that reaction-diffusion in emulsions can generate 3D Turing pattern16. In this out-of-equilibrium system, the Turing patterns exhibited by the Belousov-Zhabotinsky (BZ) reaction droplets would not exist if the reaction stopped.
Recently Philp has reported the propagation of a reaction-diffusion fronts of a replicator17. The replicator is the product of reaction between a maleimide and a nitrone modified with a fluorescent dye, and the reaction is autocatalytic because the replicator templates its own formation. The replicating process brings about a change of color in the sample. Taking advantage to this optical change, a reaction-diffusion front has been initiated and propagated when preformed replicators are seeded to a library with homogeneous precursors. The system combined replication with reaction-diffusion, demonstrating that it is possible to achieve spatially evolution of replicating species (Figure 1.4).
Figure 1.4 A propagating front driven by a synthetic replicator. Reproduced from ref 17.
1.2.5 Dissipative self-assembly
In living cells important assembly and disassembly processes of supramolecular biopolymers such as actin and microtubules are controlled by fuel molecules. As adenosine triphosphate (ATP) is the fuel for actin assembly and microtubules use guanosine triphosphate (GTP) as fuel. Stopping supply of the chemical fuels leads to function loss and apoptosis of the cells. From the aspect of thermodynamics, living systems are neither thermodynamically stable, nor kinetically trapped. Life is out of equilibrium which requires a continuous supply of energy/materials to maintain a steady state, where sometimes unpredictable functions may
emerge. Learning from biology, the field of synthetic self-assembly has expanded beyond systems at thermodynamic and kinetic control to out-of-equilibrium regimes and several systems associated to chemical fuel-to-waste conversion (so called dissipative system) have been reported.
Van Esch has presented a dissipative self-assembled system (Figure 1.5A)18. The carboxylic acid precursor does not form gels due to electrostatic repulsion, but upon methylation (using dimethyl sulfate as chemical fuel) the resulting ester can self-assemble into fibers. However, the esters are not stable but spontaneously hydrolyse back to the acid, causing the disassembly of the fibers. By controlling the addition of the chemical fuel, the lifetime of the dissipative gels can be tuned. Interestingly, fibers tend to collapse suddenly, which is similar to the catastrophic collapse of microtubules. This is an excellent example where by operating self-assembly out-of-equilibrium, advanced life-like property emerged.
The system above is implemented through batch-wise supply of the chemical fuel. However the fuel supply in natural systems is mostly continuous. Recently, Hermans has descried a flow system for dissipative self-assembly, in which ATP is added as fuel and waste is removed continuously (Figure 1.5B)19. There are two molecules that can form supramolecular polymers with different structures: PDI and p2-PDI. Peptide-perylenediimide dervative PDI can be phosphorylated on the serine by protein kinase A (PKA) to give diphosphorylated p2-PDI. The reaction is fueled by ATP, leaving ADP as waste. The backward hydrolysis of the phosphate on p2-PDI can be realized by γ-protein phosphatase (γPP), and the phosphate Pi is the waste. The assembly/disassembly of the polymers is regulated by phosphorylation and dephosphorylation. Waste products (inorganic phosphate) inhibit the reaction cycles when allowed to accumulate. However this poisonous waste was flown out, giving rise to a dissipative steady state (both polymers are present) with a long lifetime, which is otherwise unattainable. While thissystem relies on reactions by biomolecules (enzymes), it would be nice to operate self-assembly out-of-equilibrium within fully synthetic systems.
Figure 1.5 (A) Structure and assembly of synthetic molecules, which can transform to active materials through the
consumption of a chemical fuel. Reproduced from ref 18.(B) Adding fuel and removing waste continuously enables a
long-lived ouf-of-equilibrium state. Reproduced from ref 19.
1.3 Darwinian evolution in chemical systems
In his famous book On the Origin of Species, Darwin demonstrated the principles of diversification in biological systems, with complexity and diversity resulting from the evolution of life4. The concept of Darwinian evolution has been extended to chemical systems by Spiegelman in his study on the evolution of RNA20. There are three processes in Darwinian evolution: replication, mutation and selection. Species relies on replication to form a population. Mutations may occur during replication, in which natural selection may act. Correspondingly, these three elements may also be introduced into synthetic systems. At the molecular level, mutations arise during the replication of autocatalytic molecules, subsequently the competition between (parents and) mutants may lead to the survival of the fittest replicating molecules by a process of selection. Next we will discuss the state of art concerning research on replication, mutation and selection in synthetic systems, respectively (Figure 1.6).
Figure 1.6 Replication, mutation and selection: three key processes that are involved in Darwinian evolution.
1.3.1 Replication
The existence of life relies on the process of self-replication, the ability to make copies of itself. To define a replicating system, it is important to identify the relation between autocatalysis and self-replication. In a simple autocatalytic reaction, the product acts as a catalyst. An autocatalytic reaction can be referred to as replication only when information is transferred from the autocatalyst to its products.In the following, representative self-replicating systems will be briefly discussed, then contributions from our group will be presented.
1.3.1.1 Self-replicating systems
Nearly all reports on self-replication are based on the so-called minimal self-replicating system21. This classic design requires that the reaction between precursor A and B can be accelerated by template T through the generation of a ternary complex [A.B.T]. Ideally after the reaction the duplex [T.T] can dissociate and each T will mediate a further round of replication (Figure 1.7). However, usually the replication efficiency in this mechanism is poor, as the
formation of the duplex prevents T from self-replication. Two regimes in replication have been identified: parabolic and exponential. Parabolic replication refers to systems with limited efficiency due to replicator duplex formation while in exponential replication the duplex problem is overcome, allowing exponential growth of the replicator. The latter has been realized by our group recently22.
Figure 1.7 Minimal system for self-replication. Template-directed ligation of two replicator precursors A and B leads
to the formation of a termolecular complex that reacts and dissociates subsequently, releasing two free replicator molecules. The dissociation of the duplex affects the replicating rates. Reproduced from ref 21.
The first example of template-directed self-replicating involved oligonucleotides and was reported by Von Kiedrowksi23. The replicator hexadeoxynucleotide was capable of increasing the rate of its own formation, however its growth was parabolic due to product inhibition. Later, the same group (Figure 1.8A) has demonstrated the exponential growth of oligonucleotides
using an elegant technique SPREAD (Surface-Promoted Replication and Exponential Amplification of DNA analogues)24.
In 1996, Ghadiri reported the first self-replicating peptide using a 32-residue α-helical peptide based on a zipper-like sequence for recognition (Figure 1.8B)25. Subsequently Ghadiri described more complex networks based on the original design, featuring cross-catalysis26 and chiral selection27.
More recently, to capture the complexities of living systems within synthetic chemical systems, self-replication has been approached by systems chemistry28. Compared to the early studies of replicating systems that use only irreversible reactions, in the systems chemistry approach replication reactions were coupled to other reaction featuring. Philp has demonstrated the first example of self-replicating systems emerging from dynamic combinatorial libraries (Figure 1.8C)29. Two nitrones and two imines exchanged components through reversible reactions. Upon addition of maleimide four possible products can be formed but only one of them dominated as it is produced in an autocatalytic fashion.
Figure 1.8 Replicating systems involving (A) Surface-assisted replication (B) Replication based on coil-coil peptides (C) Dynamic combinatorial replicator libraries. Reproduced from ref 24, 25 and 28.
1.3.1.2 DCC and our group’s replicating system
Our group has studied emergent properties arising from dynamic molecular networks, which recently have been shown to be powerful tools for investigating self-replicating molecules. The methodology used is called Dynamic combinatorial chemistry (DCC), which is a powerful tool in systems chemistry30. A dynamic combinatorial library (DCL) is a molecular network in which library members can interconvert through reversible chemical bonds. One of the most used chemistries in DCLs is thiol-disulfide chemistry (Figure 1.9): Oxidation of thiols gives disulfides,
and disulfide exchange is active under basic conditions where disulfides can be attacked by thiolate anions. Fully oxidizing the library or operating at acidic pH effectively quenches the exchange reaction due to the lack of thiolate ions. There are various applications of DCC: synthetic receptors, catalytic systems and complex self-assembled systems. In the next section we will discuss in detail how self-replicating systems can be developed with DCC.
Figure 1.9 Oxidation of thiols to disulfides and thiol-disulfide exchange.
The most relevant building block reported by our group31 features two thiol groups for reversible thiol-disulfide chemistry and a peptide chain composed of alternating hydrophobic and hydrophilic amino acids that are prone to form β-sheets through stacking. The building block was dissolved in basic borate buffer (enabling thiol-disulfide exchange) and oxidized by air, which led to the formation of a dynamic combinatorial library of macrocycles with different sizes. The composition of the library was monitored by UPLC, and without agitation, no self-replicating species appear, allowing non-assembling 3mer and 4mer to become the dominating species. However, upon agitation (stirring or shaking), macrocycles with larger sizes emerged, which resulted in the formation of fibers, verified by TEM.
The macrocycles were proven to be self-replicators by seeding experiments. A small amount of the assembled macrocycles was added to a DCL composed of mostly 3mer and 4mer. Compared to the control sample without seeding, the seed was capable of speeding up the formation of copies of itself. This behavior can be called replication, because the information regarding ring size and composition is transferred to the newly formed replicators. We have demonstrated that the replicators can grow exponentially under the right conditions. An elongation/breakage model for this self-assembly driven self-replication has been proposed: the fibers act as templates and get elongated by the stacking of newly formed replicating macrocycles. Mechanical agitation breaks the growing fibers, therefore producing more fiber ends for further replication.
The ring size of the replicators depends on the peptide sequence, more specifically, on the interactions between the peptide chains32. With more hydrophobic peptides, the interaction between them is stronger, so a smaller number of peptide chains is sufficient to allow for the stacking of macrocycles. Instead of changing the peptide sequence, the solvent composition can also alter the interactions between the building blocks, thus dictating the ring size of the replicators33.
Notably, while emerging from a DCL, the process of replication is under kinetic control, which is a first step toward the out-of-equilibrium character of life (Figure 1.10).
Figure 1.10 Formation of a dynamic combinatorial library consisting of macrocycles, the larger of which nucleate into
fibers, which elongate and break through mechanical forces, resulting in yielding exponential growth of the replicating macrocycles.Reproduced from ref 31.
1.3.2 Mutation
The interaction between replicators and the environment is one of the key points in Darwinian evolution. A replicator that is best adapted to the local environment, might not always maintain the fittest when there are changes in external conditions. The new environment may favor specific mutants that differ from their parents. These mutants will become dominating if their rate of formation and/or resilience to destruction, exceeds that of their predecessors.
The concept of quasi-species, proposed by Eigen, has deepened scientists’ understanding of mutation in evolution34. Instead of only considering a single replicator as the unit in which evolution acts, the quasi-species (mutants) are rather a distribution around the parental replicator. The mutants in the distribution are cross-catalytic, where further mutation may happen when a selection pressure is applied.
Our group has reported an example demonstrating the diversification of self-replicating species (Figure 1.11)35. The study involved the combination of two different peptide building
blocks F and S. Individually, F formed self-replicating 6mers (F)6and S formed self-replicating 8mers (S)8. However only 6mers formed upon mixing F and S in one DCL. The obtained species can be differentiated to two sets: set 1 (F)6, (F)5(S)1, (F)4(S)2, (F)3(S)3, and (F)2(S)4, (F)1(S)5, (S)6as set 2. Set 1 acts as template and transfers the information of ring size to set 2, therefore set 1 is the ancestor of set 2. The system shows some of the features of a quasi-species, while strictly speaking, it is not, as quasi-species are a product of evolution, and the present system cannot evolve as it lacks a replicator destruction pathway.
Figure 1.11 Formation of two sets of replicators, the second replicator set is a descendant of the first set. Reproduced
from ref 35.
1.3.3 Selection and dynamic kinetic stability
Competition comes after mutation processed by natural selection. Operating natural selection at the molecular level requires the simultaneous occurrence of replication and destruction, under which conditions the species with higher accumulation rates may survive, as the weaker replicators go extinct.
Indeed, evolution of replicating species may not happen without a selection pressure. At the molecular level, simple replication reactions alone only converge to a thermodynamically stable or kinetically trapped state when molecules that constitute the food source are consumed completely. However, life is not thermodynamically controlled or kinetically trapped. Instead, life is out of equilibrium, as living systems require constant flows of matter/energy to maintain a steady state (Figure 1.12). So it is essential to operate replication in an
out-of-equilibrium regime. These considerations has led Addy Pross to coin the term ‘dynamic kinetic stability’ (DKS)36. Replicators are evolvable in DKS states where replication and destruction occur at the same time. The process of recycling of building blocks may stimulate the replicators with autocatalytic and nonlinear nature to differentiate towards multiple evolutionary possibilities, resulting in complexification. DKS may well be the key in the search for the missing link between inanimate matter and simple forms of life, as DKS is closely related to many features of life, such as the dynamic character, diversity and complexity. To achieve Darwinian evolution at the molecular level, the congruence of replication and DKS is essential, which unfortunately has not been achieved yet. Achieving such congruence is one of the aims of this thesis.
Figure 1.12 Three regimes of a chemical system: (a) thermodynamic equilibrium (b) kinetic trap (c) out-of-equilibrium
systems.
1.4 Origins of homochirality
Homochirality is one of the most striking features of life, as most molecules of life possess the same handedness. The origin of biochirality is an important topic in origin of life research, which itself is already a grand challenge. This intriguing issue is still controversial due to its inherent complexity. However, there is a consensus that the biochirality is closely link to the processes of spontaneous mirror symmetry breaking. Homochirality could have emerged following spontaneous mirror symmetry breaking by combined with additional processes, such as autocatalysis. The emphasis of this section is on studies focusing on the role of autocatalysis in the origin of homochirality.
In a classic paper published by Frank, autocatalysis has been implicated in the origin of biological homochirality, which is often considered to be the mechanism associated with the emergence of self-replicators at the stage of early life37. In the model he introduced the D and L enantiomers which are autocatalytically produced by reactions between achiral molecules A and B. The D and L enantiomers are consumed through a chiral inhibition reaction, where inactive species DL are formed. The whole system is dissipative by continuously flowing in the achiral substrates A and B and flowing out the inactive product DL by additional processes, such as sublimation, precipitation, etc. If an initial chiral imbalance is created in the system, then the corresponding enantiomer becomes amplified, as the system reaches to a stable state.
Even though Frank provided a relatively simple and efficient model for the emergence of homochirality, there was no experimental implementation until half a century later, when Soai repeated a system that could potentially follow the Frank model38. In this example of enantioenrichment, organozincs act as an alkylating reagent of achiral aldehydes. The chiral product alcohol can interact with the organozincs and enhance the rate of the reaction, thus acting as an autocatalyst. The reaction is highly sensitive to tiny initial imbalances. In one of the example, the 0.1% ee values can be amplified to 85%. More recently, Tsogoeva has reported Mannich and aldol transformations, which appear to show similar properties of autocatalysis and amplification39. Compare to the Soai reaction, these reactions operate close to prebiotically relevant conditions.
The issue of homochirality has also been investigated for two important classes of biomolecules: nucleic acids and peptides. One of the most striking examples of autocatalytic peptide replicators with chiral selection was demonstrated by Ghadiri (Figure 1.13)27. The system was based on self-replicating helical peptides. There are two enantiomeric pairs of electrophilic E and nucleophilic N peptide fragments. One pair is composed of all L amino acids, NL and EL, and the other contains only D amino acids, ND and ED. Four possible products can be obtained through the reactions between the fragments, including homochiral products TLLand TDDand heterochiral TLDand TDL. Only the homochiral TLLand TDD were autocatalytic and they did not cross-inhibit (in contrast to the situation in the Soai reaction). Moreover, peptides with one L or D substitution were incapable of autocatalysis, while they could promote the formation of homochiral species. These results suggest an important role of self-replicating peptides in the origin of biochirality.
Figure 1.13 A helical peptide system able to amplify homochiral products through self-replication. Reproduced from
ref 27.
In summary, Frank’s model provides useful guidance on how homochirality might emerge in early life, and a large number of insightful theories have been provided to expand and modify the Frank model. However, the experimental examples of autocatalytic reactions that amplify enantiomeric excess remain rare. The state of the art concerning research on autocatalysis in the context of the origin of homochirality really calls for new chemistry and methodological tools to verify ongoing theoretical research.
1.5 Aims and outline of the thesis
Self-replicating molecules play a fundamental role in the origin of life studies. The self-replicating molecules carry heritable information that is encoded in molecular structure and replicators with different molecular structure represent mutations capable of passing distinct inherited information to the next generation. During the replication process kinetic selection acts on the mutations resulting in the survival of the fittest variation, which can probably undergo Darwinian evolution leading to complexification of the replicating molecules. Currently there are no examples of using kinetic selection to enable the complexification of the replicating molecules, which is essential to the emergence of life. Using a systems chemistry approach, this thesis described a self-replicator with increasing molecular complexity under out-of-equilibrium condition. Moreover, considering the importance of chirality in origin of life and the lack of suitable system for investigation, a dynamic combinatorial chemistry-based self-replicator with chiral selection was studied.
through systems chemistry approach. As evolution is the heart of the development of life, the state of art concerning the research on Darwinian evolution at the molecular level was presented. To overcome the current challenges we introduced our group’s replicating systems as a promising platform. At last, the issues of the origin of homochirality was discussed. InChapter 2, we reported the emergence of diverse synthetic self-replicators from dynamic
combinatorial libraries (DCC) under different environmental conditions. We investigated the behavior of our peptide building block in the solutions containing different concentration of salt guanidinium chloride (GuHCl). As the concentration of GuHCl increases, the replicating species shifted from the original 6mer via intermediate 5mer to 3mer. Seeding experiments suggest that this complex network with competing replicators might be a suitable platform to explore Darwinian evolution at the molecular level.
Chapter 3 presented a kinetic study of the redox and exchange reactions in thiol-disulfide
chemistry. We measured the kinetics of redox and exchange reaction in different pH using aromatic thiols/disulfides, which is the first step towards achieving self-replication out-of-equilibrium.
Building on the previous chapters, in Chapter 4 we described the first successful
implementation of dissipative self-replication. Based on the discovery reported in Chapter 2, we determined a condition with a certain concentration of GuHCl in which one 3mer was the thermodynamically most stable replicator, while 6mer was less favored. However, we showed that thermodynamically less stable yet molecularly more complex replicator 6mer, which was also found to replicate slower than the 3mer, can be populated when the system was in an out-of-equilibrium regime through applying continuous redox reactions (i.e. a formation/destruction regime). This result represents the first experimental manifestation of Addy Pross’s concept of dynamic kinetic stability (DKS) in systems of synthetic self-replicators. In addition, the molecularly more complex 6mer is functionally more proficient in the catalysis of a model reaction.
InChapter 5, we described a self-replicator with chiral selection. Based on the results from
Chapter 2, we discovered that a replicating 5mer shows enantimeric self-sorting from a racemic mixture. The homochiral 5mer can grow only when the food source with the same chirality is provided. These results contribute towards understanding the role of autocatalysis in the origin of homochirality.
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