Steps towards de-novo life
Monreal Santiago, Guillermo
DOI:
10.33612/diss.121581426
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Monreal Santiago, G. (2020). Steps towards de-novo life: compartmentalization and feedback mechanisms in synthetic self-replicating systems. University of Groningen. https://doi.org/10.33612/diss.121581426
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Compartmentalization and feedback mechanisms in
synthetic self-replicating systems
This work was financially supported by the European Union through the Marie Skłodowska-Curie ITN fellowship "Multi-Stimuli Responsive Molecular Systems and Materials (ResMoSys)" (Grant agreement ID: 642192)
Printed by Gildeprint, The Netherlands ISBN: 978-94-034-2573-3 (printed version) ISBN: 978-94-034-2572-6 (digital version)
Front cover painting by Aaron Lawrance (aaronlawrancedrawing.tumblr.com) Back cover painting by Federico Santiago Martínez
Compartmentalization and feedback mechanisms in synthetic
self-replicating systems
PhD thesis
to obtain the degree of PhD at the University of Groningen
on the authority of the Rector Magnificus Prof. C. Wijmenga
and in accordance with the decision by the College of Deans. This thesis will be defended in public on
Friday 15 May 2020 at 11.00 hours
by
Guillermo Monreal Santiago
born on 30 November 1992Assessment Committee
Prof. dr. W.T.S. HuckProf. dr. M.M.G. Kamperman Prof. dr. T.M. Hermans
1 Introduction: Progress towards the synthesis of fully artificial ("de-novo")
life 7
1.1 Why do we research de-novo life? . . . 8
1.2 What are we trying to make? Definitions of minimal life . . . 9
1.3 Self-replication . . . 10
1.4 A synthetic replicator based on dynamic combinatorial chemistry . 14 1.5 Compartmentalization . . . 17
1.6 Metabolism . . . 21
1.7 Systems chemistry and emergent properties . . . 24
1.8 Contents of this thesis . . . 25
1.9 About the structure of this thesis . . . 26
1.10 References . . . 26
2 Mechanistic studies uncovering a new pathway for supramolecular poly-merization and replication 35 2.1 Introduction . . . 36
2.2 Results and discussion . . . 38
2.3 Conclusions . . . 43
2.4 Material and methods . . . 44
2.5 Supplementary material . . . 50
2.6 Further reading . . . 55
2.7 Acknowledgements . . . 55
2.8 References . . . 55
3 Complex coacervation as a compartmentalization strategy for synthetic self-replicators 59 3.1 Introduction . . . 60
3.2 Preliminary results . . . 62
3.3 Results and discussion . . . 64
3.4 Conclusions . . . 71
3.5 Further experiments . . . 73
3.6 Materials and methods . . . 74
3.7 Supplementary material . . . 81
3.8 Acknowledgements . . . 87
3.9 References . . . 88
4 Emergence of light-driven protometabolism upon recruitment of a pho-tocatalytic cofactor by a self-replicator 93 4.1 Introduction . . . 94
4.2 Preliminary results . . . 97 5
4.3 Results and discussion . . . 99
4.4 Conclusions . . . 104
4.5 Further experiments . . . 105
4.6 Material and methods . . . 107
4.7 Supplementary material . . . 112
4.8 Acknowledgements . . . 127
4.9 References . . . 127
5 Towards an autonomous chemical oscillator 131 5.1 Introduction . . . 132
5.2 Preliminary results . . . 134
5.3 Results and discussion . . . 136
5.4 Conclusions . . . 146
5.5 Further experiments . . . 147
5.6 Materials and methods . . . 149
5.7 Supplementary material . . . 153
5.8 Acknowledgements . . . 163
5.9 References . . . 163
6 Conclusions and perspectives 167 6.1 Conclusions and perspectives . . . 168
6.2 References . . . 172
English summary 173
Nederlandse samenvatting 175 Sumario en español 177
Acknowledgements 179
About the author 183
Introduction: Progress towards
the synthesis of fully artificial
("de-novo") life
In this chapter, I review previous research aiming at the synthesis of fully artificial life. The chapter starts with a discussion of the importance of research in this area, followed by an overview of different definitions of life - including the one that will be used in this thesis. Next, I discuss in detail the properties that are fundamental to life according to that definition (replication, compartmentalization and metabolism) and the progress to-wards their experimental realization, both individually and combined in a single system. I also address how complex chemical reaction networks can lead to emergent properties found in living systems, such as oscillations. To conclude, I introduce a synthetic replica-tor based on dynamic combinareplica-torial chemistry and explain its replication mechanism in detail. New properties will be added to this replicator through the rest of this thesis, using it as starting point towards the synthesis of de-novo life.
1.1
Why do we research de-novo life?
"How did life begin?". Or "What is the secret of life?". Even "Can we unravel the puzzle of life’s origins?". Variations of the same question keep topping the lists of "unanswered questions in science" year after year, appearing in scientific jour-nals [1], newspapers [2], books [3], scientific agendas of countries [4] and even as quests with millionaire rewards [5]. This is an indication of two features of origin-of-life research: how widespread is the interest in advancing the field, and how complicated it is to do so.
Considering that life on Earth is estimated to have emerged at least 3.5×109
years ago [6], it is naturally very difficult to understand what life was like in its early stages. Therefore, most approaches towards studying the origin of life are based on the knowledge that we do have: what life is like now. Both "top-down" [7–9] and "bottom-up" [7, 8, 10, 11] approaches ultimately aim to synthesize life based on the building blocks and chemical reactions of our current biochemistry. But there is no guarantee that all life in the universe must be based in those build-ing blocks. If life exists in other planets, it might have originated in different conditions, evolved in different ways, and therefore be completely different to the lifeforms that we can recognise today [12, 13].
Furthermore, as it has been proposed by Cleland and Copley [14], and Davies and Lineweaver [15], we do not even have any certainty that all terrestrial life is based on these building blocks and reactions. Their shadow biosphere hypothesis [14–16] argues that life might have emerged more than once on Earth, and that there is no obvious reason why only one of the original lifeforms would have survived. Moreover, since the techniques that we currently use to study micro-bial life rely on the detection of nucleic acids, they argue that organisms with a different genetic material would be completely invisible to us.
I believe that these considerations are enough to justify the research towards completely synthetic ("de-novo") life. Creating chemical systems that possess the same characteristics as life but are based on a completely different chemistry could help us think of new ways in which life could have started, update our methods to detect it, and in general change our current paradigms and the way we think about it. But in order to design life, we first need to define it: not by its chemical composition, but by its functional characteristics.
Before continuing the discussion on the functional characteristics of life, there is one last reason to synthesize life-like systems: life is useful. Concepts like self-replicating robots [17], adaptive materials [18], or synthetic chemotaxis [19] are all inspired by living systems and, beyond the implications discussed above, have or could have a number of practical applications in different areas. Life has de-veloped a number of efficient solutions to different problems - it is only natural to try to learn some lessons from it on how to solve ours.
1.2
What are we trying to make? Definitions of
minimal life
Defining life is a challenging task by itself [20]. Different definitions have been proposed over the years [21], each of them encompassing some properties of life and leaving others out. One of the most widely spread definitions is "a self-sustained chemical system capable of undergoing darwinian evolution", formulated among others by Joyce [22]. This apparently simple definition implicitly includes a number of fundamental properties of life: metabolism, information transfer, possibilities for mutation and selection, and energy dissipation. However, its crit-ics have pointed out that the life that it defines is too similar to its current form to be representative - in the event that we found systems that evolved through, for example, Lamarckian evolution, it would be hard to classify them as "not-alive" based only on that technicality [23].
A more modern definition of life has put the focus on its kinetics rather than in specific properties. According to Pross, life is "a self-sustaining kinetically stable dynamic reaction network derived from the replication reaction" [24]. In their work, Pross and his coworkers develop the concept of dynamic kinetic stability (the persistent state of replicators that emerges in conditions of reproduction and de-struction) [25] and propose how the emergence of other properties of life might be related to it [26–28]. Many other definitions of life have been proposed besides these two examples, based on various aspects such as chemical building blocks, ability to store and process information, or complexity [21].
This thesis follows the definition of minimal life proposed by Ganti in 1971 [29] and used by others since then [30]. This definition establishes that a minimal living system (a "chemoton") should possess subsystems to perform three basic functions: self-replication, metabolism, and compartmentalization.
Chemical reactions Organization Subsystems Organization (metabolism) motor Chemical (replication) system Information (compartment) separationSpatial Minimal form (Chemoton) Chemical systems
Biological systems of life
Figure 1.1: Subsystems forming the minimal unit of life (Adapted from [29]) 9
In the following sections, I will introduce each of these properties, their im-portance for living systems, and to which extent they have been realised synthet-ically. It is important to note that, besides being considered the minimal criteria for life by Ganti, these properties are related to other definitions of life too. Self-replication is a basic element of dynamic kinetic stability, and metabolism is also intimately related to it [27]. Replication is necessary for darwinian evolution, and both compartmentalization [31] and metabolism [32] can also play critical roles in it. Theoretical studies have also postulated that the combination of collective me-tabolism, transient compartmentalization, and a network of self-replicators can be a solution to the Eigen paradox - the apparent impossibility of developing a large amount of genetic information without error correcting mechanisms and vice versa [33]. This trend suggests that, perhaps, it is more important to study the synthesis of "life" according to any definition than to select a particular one: all definitions might be related to each other in ways that are not immediately evident, and therefore any advances in making life of any kind might be relevant for all of them.
1.3
Self-replication
Self-replication can be defined as the ability of a system to autonomously catalyse the formation of copies of itself, in such a way that information is transferred from each generation to the next one. This transfer of information is the mechanism through which life persists over time. All known life forms exist in an out-of-equilibrium state and are destroyed eventually, and this process is compensated by the formation of new copies that are made from different molecules but remain mostly identical to the original (see below).
From a chemical point of view, information transfer is also what distinguishes self-replication from autocatalysis. While other autocatalysts will also catalyse reactions similar to their own formation (i.e. surfactants forming micelles that in-crease the reactivity of their hydrophobic precursors by solubilizing them), self-replicators must have a certain degree of specificity [34]. Interestingly, Darwinian evolution, the motor that has driven the development of life to its current diver-sity, relies on replicators not being completely specific: errors lead to mutations and mutations lead to evolution. For replicators to evolve, their replication fi-delity must be within a certain threshold: sufficiently high for information to be transferred, but low enough for mutations to take place [35].
In the molecular level, this specificity is normally achieved through some type of templating effect. Self-replicating molecules typically interact with their pre-cursors through precise supramolecular interactions, organising them in the right configuration and catalysing the formation of a copy of themselves with the same structural information. A scheme showing a mechanism of self-replication based on a simple example of this templating process is shown in Figure 1.2a. In this
mechanism, the replicator a catalyses its own formation by binding its precursors
b and c, and preorganising them in the right configuration for their coupling to
take place. The information that is transferred is the structure of a, and the speci-ficity of the replication comes from the interaction between the binding motifs of
a, b and c. The binding between each of the precursors and the replicator must
be strong or the uncatalysed reaction between precursors will play an important role, losing the specificity that comes with the templating. However, this strong binding also leads to an undesirable side-effect: the complex a·a is also strongly bound and therefore hard to dissociate, and replicators following this mechanism tend to suffer from self-inhibition. This typically limits their order of autocataly-sis (p) and leads to parabolic, (p = 1/2) instead of exponential (p = 1) replication [36]. This is inconvenient for using them to develop models of living systems: it has been proven that p needs to be equal or larger than the order of replicators in their destruction for achieving selection and Darwinian evolution, and the lat-ter typically equals 1 [37, 38]. Several synthetic replicators, based on nucleotides [39, 40], peptides [41], ribozyme fragments [42–44], and completely synthetic mo-lecules [45], follow mechanisms similar to the one of Figure 1.2a - some examples are shown in Figure 1.3.
a
b c
a·b·c a·a
(a) Self-replication based on the templated formation of a dimer.
(b) Self-replication based on the templated formation of a supramolecular polymer
Figure 1.2: Different mechanisms of self-replication that have been achieved with syn-thetic systems. The self-replicator shown in (a) forms only discrete structures and needs to be free to template its own formation, therefore it is inhibited by the formation of the dimer a·a. The self-replicator shown in (b) templates its own formation while still bound to another template, so it self-assembles forming a supramolecular polymer. The elongation-breakage mechanism shown here can lead to exponential growth. See below for examples of both types of systems.
O O N HN NH N O N H N N O N O OH O O N3 O O O O N HN NH N O N N N O O O O N3 O N N O O O O N HN NH N O N N N N O N HN NH N O O O O N N H O N HN NH N O HO O O H H H H H H H H N H (a) (b) NCL (c)
Figure 1.3: Examples of synthetic replicators based on different chemistries but follow-ing the templatfollow-ing principle shown in Figure 1.2a. (a) A self-replicator based on synthetic structures that react through an azide - maleimide cycloaddition, adapted from [45]. (b) A self-replicating ribozyme that ligates its precursors through a 3’,5’-phosphodiester bond, adapted from [40]. (c) A self-replicating peptide that catalyses amide bond formation be-tween two peptide fragments (one of them modified with a thiobenzyl ester) by native chemical ligation (NCL). Adapted from [42]. See the original sources for the detailed struc-tures.
The mechanism shown in Figure 1.2b is based on a similar templating effect (each trimer organises its three precursors in the same configuration as itself), but it solves the self-inhibition problem: instead of requiring the newly formed molecule to be dissociated in order to act as a template, each of the trimers at the end of the stack can act as a template while still bound to the others. This leads to the formation of non-discrete structures (the figure shows 1D fibres but other struc-tures are possible [46, 47]), that can achieve exponential replication in a growth-breakage regime [48]. Different synthetic systems follow this mechanism [46–50] (Figure 1.4), including the one that is central to the work of this Thesis (see Section 1.4).
+
1-mers
plates fibrils nanotubes
Ligation reaction
Templating and autocatalysis
Self-assembly
(a) Self-replication based on the formation of an amphiphilic peptide through NCL
(b) Self-replication based on the formation of a tetramer that self-assembles into a 2D-sheet
Figure 1.4: Examples of self-replicators based on the formation of non-discrete structures. In (a) the self-replicator is a peptide formed through native chemical ligation (NCL). This peptide self-assembles in transient β-sheet structures that catalyse the native chemical lig-ation of its precursors by templlig-ation [50]. In (b) the self-replicating structure is a tetrameric disulfide formed upon oxidation of an aromatic dithiol. The tetramer is originally formed upon addition of a trigger that acts as a template, but once that it has surpassed a crit-ical aggregation concentration it self-assembles into sheets that catalyse the formation of more non-templated tetramers [46]. For another example of a disulfide-based non-discrete replicator (on which the work described in this thesis is based), see Section 1.4.
Let me comment on these two mechanisms of self-replication and their prebi-otic relevance. We can argue that replication in known current living systems is based on a mechanism analogous to the one shown in Figure 1.2a: behind all the added complexity and the enzymes that contribute to the process, information is transferred from one DNA chain to another through the binding of nucleobases to a template. Just as in simpler examples of this mechanism, DNA then forms a dimer that needs to be dissociated for replication to take place [51]. Despite this being the case now, recent theories argue that in a prebiotic context the mecha-nism shown in Figure 1.2b might have played an important role too. Amyloid peptides have been proposed as a self-replicator that predates nucleic acids, due to their stability, information-transfer capabilities and relative simplicity [52, 53]. Experimental evidence of self-replication in amyloids has been found, too [54]. This again highlights the importance of researching different mechanisms for the emergence of functional characteristics of life: there are, at least, two models of prebiotic self-replication based on substantially different principles.
1.4
A synthetic replicator based on dynamic
combinatorial chemistry
A tool to develop large networks of reactions from relatively simple building blocks is dynamic combinatorial chemistry (DCC) [55, 56]. DCC is based on the formation of reversible covalent bonds that are spontaneously formed and destroyed in mild conditions - called dynamic bonds. By mixing one or more building blocks based on these bonds and allowing them to react reversibly, a dy-namic combinatorial library (DCL) is formed, which can contain a large number of components even if it was made from few building blocks. Since the reactions converting library members into one another are reversible, DCLs are normally considered to be in thermodynamic equilibrium, settling into a state with min-imum energy. Furthermore, this also makes DCLs responsive: for example, if a member of the library is bound to an external template or self-assembles into a structure, its stability increases, the equilibrium shifts towards its production, and its concentration is amplified.
Several dynamic bonds have been discovered [57] and applied to fields rang-ing from the development of protein inhibitors [58] to responsive polymers [55]. The DCLs that will be studied in this thesis are based on disulfide bonds. Disul-fide exchange takes place in mild basic conditions in presence of catalytic amounts of thiolate [59], and has been used in DCC since 2000 [60, 61]. DCLs of disulfides are readily formed upon oxidation of the corresponding thiols in buffered aque-ous solutions (Figure 1.5), and can be "frozen" (their exchange can be stopped) by changing the pH to acidic and therefore protonating the thiolates.
RS SR 2 x RSH [ox] 2 x RS RS SR R'S R'S SR RS I) II) ( (
Figure 1.5: Scheme showing the formation of a dynamic combinatorial library based on disulfide exchange. (I) Oxidation of thiols to disulfides at basic pH. The oxidant [ox] can
be O2 dissolved in the solvent, or any other suitable species. (II) Mechanism of disulfide
exchange. A catalytic amount of thiolate can attack disulfide bonds, releasing another thiolate that can react further with other disulfides, typically leading to equilibration of the library.
Our group has developed a family of self-replicators that emerge from DCLs based on disulfide bonds [49], and studied them extensively in the last decade -observing life-like behaviours such as adaptation to different environments [62], diversification [63], history-dependent composition [64, 65], and parasitism [66]. These replicators are based on building blocks consisting on an aromatic dithiol and a peptide chain that alternates hydrophilic and hydrophobic amino acids
(Figure 1.6a)1. Upon oxidation of these building blocks in aqueous solutions
(typically borate buffer, pH 8.2), they form a library of disulfide-based macro-cycles that are in exchange with each other. Macromacro-cycles of a large enough size stack on top of each other, forming supramolecular fibres (Figures 1.6b and 1.6e). The self-assembly of these fibres is driven by two factors: the hydrophobicity of the aromatic core and the tendency of the peptide chains to stack in β-sheet-like structures [68]. As discussed above, the formation of these fibres causes the amplification of the macrocycle that self-assembles at the expense of the rest of the members of the library, until practically all the material is transformed into stacked macrocycles (Figure 1.6c). The sigmoidal growth curve of the fibre-forming macrocycle suggests autocatalysis, and this is confirmed by seeding ex-periments (Figure 1.6d): as expected in an autocatalytic system, the formation of the self-assembling macrocycle is catalysed upon addition of a small amount of it-self. Sigmoidal growth is only observed in stirred or shaken libraries, suggesting a replication mechanism based on growth and fragmentation. In this mechanism, the fibres grow from their fibre ends consuming the other members of the disul-fide library. Mechanical force breaks the fibres that are long enough, freeing more fibre ends and therefore accelerating growth (Figure 1.6f). Kinetic studies have confirmed this mechanism, and determined that it leads to exponential growth [48]. Interestingly, mechanical agitation is only necessary at early stages of repli-cation: if enough fibre ends are available (such as in the seeding experiment of Figure 1.6d), stirring does not affect growth rate. A new element of this replica-tion mechanism is described in Chapter 2.
1The building block represented in Figure 1.6a for R = benzyl, which will be referred to as 1 for
the rest of this thesis, is shown in the rest of Figure 1.6 as an example of this type of building blocks. We have explored related compounds with different amino acids in the fourth position, and observed similar behaviours, typically varying only in the ring size of the self-replicating species [67].
HS SH O N H H N O N H O O H N O N H R O O 1 : R=
(a) Structure of 1 and related building blocks 13 14 16 1 self-replication oxidation
(b) General scheme of the formation of the self-replicator 16
16 1 13 14 12 Time (days) 0 5 10 15 0 20 40 60 80 100 Fr acti on of [ 1n ] (%)
(c) Time evolution of libraries of 1
Seeded, stirred Seeded, not stirred Not seeded R elativ e [ 16 ] (%) 0 20 40 60 80 100 0 5 10 15 Time (days)
(d) Seeding experiment using 16
200 nm
(e) Cryo-TEM of 16fibres
Fragmentation
Growth Growth
(f) Self-replication mechanism of 16fibres, based on growth and fragmentation
Figure 1.6: Self-replication of 16and related building blocks. (a) General structure of the
building blocks that generate peptide-based replicators of the type of 16. Most of this thesis
will be related to 1 (R = benzyl). (b) Formation of 16 from 1. Upon oxidation, 1 forms a
library of disulfide-based macrocycles (mainly 13/14) in exchange with each other. One
of those macrocycles, 16, self-assembles forming supramolecular fibres that catalyse their
own formation. (c) Evolution over time of a library of 1, stirred at room temperature and studied by UPLC analysis. The relative concentrations (in 1 units) of the main components of the library are represented. Besides 1 and the linear 12, all other species are cyclic. (d)
Proof of autocatalysis of 16. The growth of 16 from a library containing mostly 13/14 is
much faster upon seeding with 5 % of 16 (purple arrow at 6 days) than without seed.
(e) Structure of 16 fibres, as studied by cryo-TEM. Detailed analysis shows fibres with
a width of roughly 3 nm, that appear both individually and associated forming twisted pairs. (f) Schematic representation of the self-replication of 16 in presence of 13/14 and
mechanical agitation. The fibres grow upon conversion of 13/14to more 16, and fragment
as an effect of the mechanical force liberating more fibre ends. This mechanism leads to exponential replication (order in replicator = 1) [48]. Panels (c) and (d) are adapted from [67]. Experimental conditions: [1] = 3.8 mM, stirring rate = 1200 rpm, buffer = 50 mM borate, pH = 8.2
1.5
Compartmentalization
The importance of compartmentalization in living systems cannot be understated. As Ganti describes [29], life is based on "soft systems" that have a maximum range in which they can be coupled to each other. As we have seen above, processes such as replication are based on intermolecular interactions and, therefore, need a minimal concentration of the relevant molecules to take place. In order to achieve a concentration that is high enough, the easiest solution is to keep the molecu-les necessary for the other subsystems inside of a boundary, where they cannot diffuse away. This function of compartmentalization must have been especially relevant in prebiotic scenarios, where the life-forming molecules were scarce and the reactions synthesizing them were primitive.
If we consider life as an open system, constantly taking up material from its environment and producing waste, another role of compartmentalization be-comes obvious: the boundary must be permeable to those molecules, allowing for the uptake of new material and preventing the accumulation of waste. Therefore, both the compartments found in canonical biochemistry and the ones proposed for prebiotic systems possess a certain selective permeability, keeping the essential components inside but allowing for the exchange of other molecules.
Compartmentalization also plays a role in more "advanced" processes related to life: without a distinct separation between different species, it is impossible to imagine selection or Darwinian evolution. Furthermore, theoretical [69] and ex-perimental [70, 71] results have shown how compartmentalization of different re-plicators greatly improves the survivability of ecosystems that develop parasites - entities that require a resource to survive, but do not contribute to its production (Figure 1.7a).
All life forms that we know rely on lipid bilayers as their main form of com-partmentalization. This is one of the reasons why most of the theories about the origin of life assume that membranes of this type must have also played a role in early stages of life, however, it is not the only one: the hydrophobic environ-ment of bilayers can be of importance for prebiotically relevant processes such as photosynthesis or electron/proton transport [72]. In any case, the membranes of current cells are based on a mixture of phospholipids with proteins and other lipids [51] - a structure so complex and dependent on the current metabolic path-ways that the general consensus is that it was developed late in the evolutionary timeline. Therefore, theories of the origin of life normally postulate that prebiotic compartmentalization must have been based on simpler amphiphiles [72–76], or on completely different building blocks (see below).
Combining compartmentalization with other functions of living systems in order to synthesize protocells is one of the main challenges of the field of artifi-cial life [77–79]. This challenge goes beyond "simply" enclosing replicators and a metabolic network inside of a compartment: in order to keep the concentration
inside of the protocells constant, the compartment must also grow and divide as the replicator does.
The growth of compartments based on lipid amphiphiles through physical autocatalysis was achieved in the 90s and extensively described by Luisi and oth-ers [80] (Figure 1.7b). Their studies already uncovered the lack of two necessary processes in this type of autocatalysis: a division mechanism for the formed
com-partments2and a coupling between the autocatalysis rates of the
compartment-forming molecules and the replicators contained in them.
(a) Compartmentalization prevents "death" of a replicating system that develops parasites
(b) Compartment reproduction by physical autocatalysis
Figure 1.7: (a) Replication network and its evolution over time of a system where the RNA replicase Qβ catalyses the formation of RNAs that can and cannot form more Qβ by trans-lation. These RNAs are named "host" and "parasite", respectively. When the system was subjected to series of incubation and dilution in bulk solution, the accumulation of parasite led to an irreversible decrease of the concentration of Qβ. However, when the experiment was repeated in a water-in-oil emulsion, the concentration of Qβ eventually recovered and led to oscillations of both host and parasite. Reproduced from [70]. (b) Physical auto-catalysis can lead to growth and division of compartments formed from a surfactant, S. This scheme represents how a precursor P, poorly soluble in the solvent used, is incorpo-rated in the structure of a compartment and then reacts to form more S. Theoretically this can lead to a bigger and less stable compartment that then divides, but for vesicles, this normally requires an additional energy input or other process. The structure of possible compartments made from S is drawn too. Reproduced from [80].
2It was initially observed that in some conditions the size of the formed compartments was
con-served after their growth, which hinted at division taking place - the so-called "matrix effect"[80]. How-ever, the exact mechanism of this process has not yet been uncovered.
The problem of compartment division is typically solved in synthetic systems through mechanical force [81] or temperature cycles [31]. Recently, new studies have shown that budding and division in lipid membranes can be triggered by the incorporation of other components in them, such as DNA [82, 83] or different lipids [84]. In these cases, the division is triggered by the change of the rigidity of the membrane in the areas where the different material is being incorporated.
The coupling of self-replication and compartment growth has not yet been completely achieved, although there has been some progress towards it. Dif-ferent groups have shown how the incorporation of nucleic acids [82, 83] (Fig-ure 1.8a) or small peptides [85] (Fig(Fig-ure 1.8b) in lipid membranes can lead to the growth of the membrane (and in some cases, as discussed above, to its division). In these examples, however, the nucleic acid or peptide that triggers the growth of the compartment is not self-replicating, and it requires an external catalyst. The work of Devaraj [86] elegantly addresses this limitation. In it, a copper catalyst catalyses both its own formation and the formation of phospholipids, leading to the continuous growth of membranes (Figure 1.8c). Both reactions are based on the alkyne-azide click reaction, which leads to the formation of a ligand that is part of the catalyst and to the addition of a second tail to a surfactant.
Besides amphiphilic lipids, other materials have been proposed for the syn-thesis of protocells. Among others, polymers, sugar-containing dendrimers, pep-tides, inorganic particles and coacervates have been used in this context [76, 79, 87]. Coacervates ocupy a special place on this list due to how some of their properties correlate to functions that are essential to life [88]. Briefly, coacer-vates are formed by liquid-liquid phase separation upon mixing of charged poly-or oligomers. Their highly charged nature efficiently concentrates charged so-lutes, and since they are mostly aqueous in nature, the structure and function of biologically relevant molecules is usually preserved inside of them. The ma-terial compartmentalized in them is in equilibrium with the external solution, but their permeability is different for different molecules, depending typically on their size and charge. Coacervates, and their ability to encapsulate a synthetic self-replicator, will be discussed in more detail in Chapter 3.
(a) Coupling of compartment growth with DNA replication
(b) Coupling of compartment growth with the synthesis of a dipeptide
(I) (II)
(c) Coupling of compartment growth with the autocatalysis of an amphiphilic catalyst
Figure 1.8: Progress towards the coupling of compartment growth and self-replication. (a) Encapsulated DNA chains replicate through PCR and accumulate in pockets of a phos-pholipid bilayer, together with an organocatalyst (green) and a precursor (V*, red) that react to form an amphiphile (V) and a side-product (E). The membrane then grows using the newly formed amphiphile and divides. Adapted from [82]. (b) Encapsulation in a lipid vesicle enhances the formation of a dipeptide (inset) that accumulates in the membrane. The vesicles containing peptide (red) can then grow by taking lipids from other vesicles (grey), that shrink. Adapted from [85]. (c) An amphiphilic catalyst (green) catalyses both its own formation (I) and the formation of a phospholipid (II) by alkyne-azide click reac-tions. When lipid bilayers containing the catalyst are mixed with azide, alkyne lysolipid,
1.6
Metabolism
The last essential subsystem of minimal life according to Ganti’s definition is what he calls a "chemical motor": a subsystem that performs chemical work. This subsystem consists on a series of reactions, collectively called metabolism, that serve several functions. First, they supply the chemicals that are necessary for the other subsystems: they produce the precursors of the replicator and the com-partment. It is evident that these precursors are not abundant in the environment of current living systems, and we can imagine the same to be true for early stages of life or synthetic versions of it. Therefore, in order for replication and compart-mentalization to work, life must develop its own chemical routes and transform the chemicals that it has access to into the compounds that it needs.
Another function of metabolism appears when we examine the nature of these compounds: they are complex molecules, typically lower in entropy than the molecules from the environment. Thus, metabolism is also a way of regulating the energy of the system: its reactions absorb energy from an external source and use it to synthesize the necessary products through endergonic reactions. This function is also related to the dissipative nature of life that was mentioned above: from a thermodynamic perspective, the energy absorbed by metabolism is necessary to keep life out of equilibrium.
In canonical biochemistry, even a single cell needs many metabolic reactions, that are catalysed by specialized enzymes [51]. However, it can be expected that in minimal systems the number of reactions will be smaller and limited to the minimal functions mentioned above. Furthermore, due to the complexity of en-zymes, it appears logical that the original metabolic reactions were catalysed by something else.
There are different possibilities regarding the nature of these original catalysts. First, they could have belonged to some of the other two subsystems, which in this case would perform a double function. Second, they could be molecules or structures otherwise not related to life, such as minerals or dissolved metal ions. Third, a combination of the other two: the catalysts might have been external molecules that were then incorporated into the living system, where their activ-ity was enhanced or modulated by the other subsystems. Examples of the first type of catalysts could be RNA ribozymes, a class of compounds able to act both as self-replicators and as catalysts for other reactions [89]. The second type of catalyst appears in theories such as the "iron-sulfur" world by Wachtershauser [90], in which the first metabolic reactions take place on mineral surfaces. Other results support this type of catalysis, such as the appearance of "metabolic" cycles in a simulated prebiotic ocean, driven by metal ions and changes in tempera-ture [91]. The third type of catalysis, the recruitment of small molecules (usually called cofactors) by early living systems is normally included as part of other the-ories [72, 89, 92]. It is strongly supported by the fact that a number of biological
processes still rely on cofactors [51]. An example of one of these processes, both essential to current life and impossible without cofactors, is photosynthesis [93].
There are not many examples of systems coupling synthetic self-replication with the catalysis of other reactions, even less with reactions that are relevant to the self-replicating entity. Rebek et al. reported a replicator of the type of Figure 1.2a that contained an imidazolidinone moiety, which led to organocatalysis of hydride-mediated reductions and Friedel-Crafts alkylations [94]. However, the catalytic reactions cannot be coupled to or combined with replication, as they happen in different conditions: catalysis takes place in 0 °C dichloromethane or chloroform, while replication needs hot benzene (Figure 1.9a). Nghe et al. have showed that combining metabolism and replication is possible for a system based on self-replicating RNA ribozymes [95]. In their system, the ribozyme is able to catalyse the deprotection of modified RNA fragments, and then use those frag-ments to replicate (Figure 1.9b). In this case, both reactions are based on phos-phodiester chemistry. Finally, our group has recently reported the catalysis of
different reactions by the disulfide-based, peptide-containing replicator 163. The
first class of these reactions is based on amine catalysis. The self-assembled su-pramolecular structure of the replicator causes the organization of some of its peptide chains into "active sites", where the lysine residues become deprotonated and available for the catalysis of retro-aldol and Fmoc- deprotection reactions (Figure 1.9c) [96]. While the retro-aldol reaction does not affect the chemistry of the replicator, Fmoc- deprotection has an important consequence: it liberates dibenzofulvene, which speeds up the oxidation of a dithiol monomer (1) to the
disulfide macrocycles 13/14. These disulfides are the precursors of the replicator,
so this process results in a faster replication as well. The second class of
reac-tions that the replicator 16catalyses, which are based on cofactor recruitment and
photocatalysis [97], will be discussed extensively in Chapter 4.
Combinations of metabolism and compartmentalization are normally focused on the encapsulation of biologically-relevant reaction networks, with impressive results such as triggering responses in bacteria as if they were in presence of a biological metabolism [98]. Other efforts go in the direction of encapsulating pre-biotically relevant cofactors in membranes [99], or catalysing the formation of compartments by reactions with a metabolic function, such as the use of energy absorbed from light [100].
To date, the combination of replication, compartmentalization, and metabo-lism in a single synthetic system has not been achieved.
3See Section 1.4 for the structure of the building block 1 and a detailed explanation of the
O t-Bu t-Bu N N N NH2 NH2 O t-Bu t-Bu H O N N N NH2 NH2 N H N O NH O O C6H6,70 °C NH O O H2N O BnHN + O Me N N H EtO O OEt O CDCl3, CSA, 0 °C O NMe O CD2CL2, DCl (cat), 0 °C
(a) Self-replication and organocatalysis by the same structure.
(b) Coupled self-replication and catalysis for a RNA ribozyme. oxidation 1 13/14 16 MeO OH O MeO O O O HN O OH O H3N O O CO2 + +
(c) Coupled self-replication and lysine-mediated
catalysis for the 16replicator.
Figure 1.9: Combinations of self-replication and catalysis of other reactions. In (a) (adapted from [94]), an organic self-replicator can act both as an autocatalyst and as a catalyst for Friedel-Crafts acylations and hydride transfer. Notice the different conditions for each of the reactions. Scheme (b) (reproduced from [95]) represents how the Azoarcus ribozyme ("WXYZ") can catalyse the release of its fragments WXY and Z by cleaving small RNA fragments ("catabolism"), and its own formation from those fragments ("an-abolism"/"autocatalysis"). Scheme (c) shows a simplified version of the self-replication
mechanism of 16 (described in detail in Section 1.4), and two reactions that can be
1.7
Systems chemistry and emergent properties
As the previous sections show, life, even reduced to its most simplified features, requires a number of reactions to be interconnected and affect each other. The study of systems of reactions exceeds the principles of traditional chemistry, that focuses on individual molecules and reactions. A new field, called systems chem-istry [101, 102], has broken with this tradition, putting its focus on networks of chemical reactions and on their emergent properties - properties that appear as a consequence of the system and that cannot be attributed to any of its components individually.
This field has researched systems that exhibit complex behaviours [103], such as oscillations and formation of patterns [104, 105]. These properties are inti-mately related to living systems too. For example, both temporal and spatial oscillations are often observed in biological processes such as gene expression, cell signaling [106–108], and as discussed before, population dynamics [70]. As in living systems, these properties are linked to energy dissipation: emergent properties come from both the complexity and the openness of chemical systems [109].
For a deeper discussion on the use of open chemical reaction networks to synthesize oscillators, and the design of one of based on the replicator described in Section 1.4, see Chapter 5.
1.8
Contents of this thesis
The aim of this thesis is to bridge the gap between synthetic chemical networks and living organisms, by implementing properties that have been traditionally associated with the latter in the former. The work described in this thesis is based on previously described self-replicating molecules that catalyse their own forma-tion through the formaforma-tion of self-assembled fibres. This thesis first studies the mechanisms behind the autocatalysis of these self-replicators, and then makes them part of increasingly complex systems by adding new components to them. The resulting reaction networks possess unique properties, described here for the first time in non-biological systems, that are then used to design new emergent behaviours.
Specifically, the work presented in this thesis uses the peptide-based macro-cycles described in Section 1.4. In Chapter 2, we use evidence from high-speed Atomic Force Microscopy to obtain new insight into the replication process. Our data shows that, in this case, self-replication is based on a novel mechanism of supramolecular polymerization. In this mechanism, the precursors of the macro-cycle that polymerises aggregate on the surface of the polymer. This creates a local high concentration that can compensate for the entropic penalty of making large macrocycles for the polymerization. The material then diffuses along the polymer chain, facilitating the process of finding a fibre end to grow from.
In Chapter 3, we use complex coacervation to compartmentalize these repli-cators. Their encapsulation into coacervate droplets is very efficient due to their high charge density, but the crowded environment of the coacervate phase al-ters their behaviour. Specifically, it changes the stability of the different library members, preventing the replication of the component that would dominate the library in solution and causing the emergence of a different replicator. In this way, we not only show the first example of self-replication inside of coacervates, but also that different replicators can emerge from the same building block as an effect of compartmentalization.
Chapter 4 describes the combination of a protometabolic cycle with replica-tion. By adding a photosensitizer to the aforementioned self-replicating system, we can couple self-replication with the transformation of inert material into the precursors of the replicator. This leads to the first synthetic example of a molecule that is able to simultaneously make copies of itself and use an external energy source to synthesize its own starting materials. We expect the efficiency of this process to change when replicators with different structures are used, potentially enabling us to achieve adaptation or selection between different replicators.
Chapter 5 explores the potential introduced by the feedback loop described in Chapter 4 for achieving autonomous oscillations. The photocatalytic reaction described in the previous chapter allows us to design a system with positive and negative feedback loops, and a delay between them. Kinetic modelling confirms
that these elements combined can lead to oscillations (although only damped ones), and provides insight into how to change different parameters of the system to reach them experimentally.
Finally, Chapter 6 provides an overview of the thesis, reflects on the progress that has been made towards the combination of life-like properties into a single synthetic system, and provides an outlook on the future possibilities of this field.
1.9
About the structure of this thesis
During the course of the PhD in which this research was performed, the long-term goals for each of the projects remained constant. However, the strategies and milestones to achieve them changed often, as research uncovered new data, contradicted our initial hypotheses and expectations, and pointed us in different directions.
A different process took place once that the main objectives were achieved. Finishing the main research lines in this thesis also opened new ones, that were only tentatively explored (if at all) due to the limited time available during the course of a PhD project.
I believe that these two scenarios are very common in research, and to a large extent intrinsic to it. Despite understanding that the main focus should be on the finished projects, this thesis would not be complete without describing those initial and final experiments.
For that reason, some chapters include sections of Preliminary results and Fur-ther experiments. The data shown in those sections should not be interpreted as a full characterization of the phenomena described on them, only as an explanation of why those strategies were not pursued at the time - or why they have enough potential to be pursued in the future.
1.10
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Mechanistic studies
uncovering a new pathway for
supramolecular
polymerization and replication
In this chapter, we use evidence from high speed atomic force microscopy and molecular dynamics to gain insight into the self-replication process of peptide-based macrocycles. In doing so, we discover a novel mechanism for supramolecular polymerization, involv-ing the accumulation of precursors on the side of the supramolecular polymer, and their subsequent diffusion to the growing ends. We confirm this pathway with kinetic mea-surements, and discover the existence of another state of the precursors: an off-pathway assembly separated from the fibres that does not contribute to the polymerization. This mechanism, which bears some resemblance to previous findings in the growth of amyloid proteins, explains (at least partially) the role of the self-assembled structures of the pep-tide replicators in catalysing their own formation, and it can have implications for the autocatalytic growth of other supramolecular structures.
This chapter is part of a submitted article: Maity, S.; Ottelé, J.; Monreal Santiago, G.; Frederix, P. W.; Kroon, P. C.; Markovitch, O.; Stuart, M. C. A.; Marrink, S. J.; Otto, S.; Roos, W. H. Caught in the act: Mechanism of supramolecular polymerization driven self-replication visualized in real time. Submitted.
2.1
Introduction
Self-assembled supramolecular structures, such as the ones formed from 16, have
gained importance in fields from materials science to biomedicine [1–3]. Since they are based on weak intermolecular interactions and not on covalent bonds, they have historically been regarded as structures in thermodynamic equilibrium. However, it is becoming more and more evident than the formation of supramo-lecular structures can also be governed by their assembly pathway and not only by thermodynamics [4–6]. Furthermore, the focus of self-assembly research is ex-panding to out-of-equilibrium systems, where the outcome of the self-assembly is undoubtedly influenced by its mechanism [7, 8].
Despite this growing interest in understanding the mechanisms behind self-assembly, the tools that we have available for their study are limited. Current methods for real-time visualization of such systems use confocal laser scanning microscopy [9] or stochastic optical reconstruction microscopy [10], but those re-quire the use of fluorescent probes and only provide resolutions down to tens of nanometers. Electron microscopy could provide higher resolutions, but its appli-cations to real-time visualization are still in their early days [11], presenting lim-itations due to the low temperature, cell morphology, and high-energy electrons that are required by the technique. Recent advances in the field of atomic force microscopy (AFM) have enabled studying dynamic processes of (bio)molecular systems using High-Speed AFM (HS-AFM) [12] at even smaller length scales, including the configurational dynamics of proteinaceous structures, [13–15] the assembly of amyloid-like fibrils [7, 16], and the movement of synthetic molecular transporters [17] with unprecedented spatiotemporal resolution. HS-AFM allows for the continuous observation of samples at room temperature and in solution, with the only constraint being that one of the components needs to be attached to a surface so its position remains constant.
In this chapter, we use HS-AFM to study the mechanism of formation of the self-replicating fibres introduced in Chapter 1. These fibres emerge sponta-neously from stirred aqueous solutions of the monomer 1. First, the thiol groups in 1 are oxidized to disulfides, giving rise to an array of small macrocycles (mainly
13/14), continuously exchanging with each other. From this library of
macrocy-cles emerges 16, which initially forms in low concentrations but has the ability
to stack into supramolecular fibres. These fibres draw the equilibrium towards
their own growth, consuming 13/14 and forming more 16 until it becomes the
main component of the library (Figures 2.1a and 2.1b). The growth mechanism of these fibres is particularly complicated to elucidate, since it involves both the
conversion of 13/14 precursors to 16 and the self-assembly of 16 into fibres. We
have previously established that it is an autocatalytic process [18], but we only understand it kinetically, not mechanistically. In this chapter, HS-AFM gives us new insight into this process, by allowing us to directly visualize the growth of
