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The systems perspective at the crossroads between chemistry and biology Andrés de la Escosura, Carlos Briones, Kepa Ruiz-Mirazo

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Received date: 25 March 2015 Accepted date: 26 April 2015 Cite this article as: Andrés de la Escosura, Carlos Briones, Kepa Ruiz-Mirazo, The systems perspective at the crossroads between chemistry and biology, Journal of Theoretical Biology, http://dx.doi.org/10.1016/j.jtbi.2015.04.036 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The systems perspective at the crossroads between chemistry and biology Andrés de la Escosura,a Carlos Briones b and Kepa Ruiz-Mirazo c,d,* a

Department of Organic Chemistry, Universidad Autónoma de Madrid, Spain Department of Molecular Evolution, Centro de Astrobiología (CSIC–INTA, Associated to the NASA Astrobiology Institute), Spain c Department of Logic and Philosophy of Science, University of the Basque Country, Spain d Biophysics Unit (CSIC, UPV/EHU), Spain b

* Corresponding author: [email protected]

ABSTRACT ‘Systemic’ views are not new in science. During the last century a number of authors pointed to the inherently systemic and dynamic nature of the living, yet their message was largely ignored by the mainstream of the scientific community. Tibor Ganti was one of those early pioneers, proposing a theoretical framework to understand the living principles in terms of chemical transformation cycles and their coupling. The turn of the century then brought with it a novel ‘systems’ paradigm, which shined light on all that previous work and carried many implications for the way we conceive of chemical and biological complexity today. In this article tribute is paid to some of those seminal contributions, highlighting the importance of adopting a systems view in present chemistry, particularly if plausible mechanisms of chemical evolution toward the first living entities want to be unraveled. We examine and put in perspective recent discoveries in the emerging subfield of ‘prebiotic systems chemistry’, reaching the conclusion that the functional coupling of protocellular subsystems (i.e., protometabolism, protogenome and membrane compartment) is the most challenging target to make qualitative advances in the problem of the origins of life. For the longawaited goal of assembling an autonomous protocell from its most basic molecular building blocks, we further suggest that a systems integrative strategy should be considered from the earliest synthetic steps, opening the way to biogenesis. KEYWORDS Origins of Life, Prebiotic Systems Chemistry, Protocells, Functional Integration, Synthetic Biology. HIGHLIGHTS • Brief historical account of the development of systemic approaches in biology and chemistry. • Review of major findings in the subfield of ‘prebiotic systems chemistry’. • Description of a central open problem: the functional integration of reacting chemical species. 1

1. Introduction Over the past decade, so-called ‘systemic approaches’ have proliferated in science, particularly in biology and chemistry, as new avenues of research that try to complement the scientific knowledge gathered to date, achieved to a large extent through reductionist methodologies. In contrast to physics, a field whose development was and is still based on the clever abstraction, isolation, modeling and measurement of relatively simple systems, chemistry and biology have huge challenges ahead that require dealing with intrinsically complex systems: systems that are complex not only in terms of the diversity of components making them up, but also of the diversity of dynamic processes and molecular interactions and transformations involved. From a general perspective, anything could be considered ‘a system’. In physics, for instance, it does not make much sense to speak of ‘systemic approaches’, because that is all it is about: explaining the properties and dynamic behavior of diverse systems, as a result of the fundamental interactions among their elementary material parts. In some areas of chemistry and biology, however, it has become meaningful to use that terminology, because the word ‘system’ or ‘systemic’ refers precisely to that still unaccomplished task of understanding intrinsically complex systems, of giving a sufficiently detailed and coherent account of their dynamic behavior in terms of their basic constituents and interactions. Quite a few scientists and philosophers of the last century were fully aware that this task is of capital importance for the advance of science and knowledge in general, but their message was largely ignored by the scientific community, most strikingly by an ample majority of biologists. One of the reasons behind could be that the methods required to face the challenge scientifically were not available at that stage yet. An obvious name to mention here is von Bertalanffy, who introduced a ‘general theory of systems’ already in the fifties and sixties (von Bertalanffy, 1950; 1968). Although this author was pointing in the right conceptual direction and making use of adequate intuitions, stemming from his previous work on the physiology of developmental systems (von Bertalanffy, 1933), he could not provide a suitable methodological toolkit to put in practice a scientific research program around such a theory. Later on, related ideas coming from thermodynamics, cybernetics and control theory were key for the elaboration of the first models that addressed the living cell’s metabolic complexity and robustness upfront, establishing a way to connect systemic variables with local, molecular ones, and demonstrating that biosynthetic pathways operate under ‘shared control’ conditions (Kacser and Burns, 1973; Heinrich and Rapoport, 1974; Savageau, 1976); nevertheless, their scope and impact remained quite limited (until many years later -- see: (Fell, 1997)). Meanwhile, the development of far-from-equilibrium 2

thermodynamics was generating very important insights about self-organization processes, relevant both for chemistry and biology (Nicolis and Prigogine, 1977; Prigogine, 1980), but as a general framework to make sense of those processes, rather than an accurate bridge between molecular specificities and macro- or mesoscopic behavior. In addition, several authors and schools of thinking in theoretical biology (e.g.: Rashevsky (1948) and Rosen (1958; 1991); Ganti, (1975; 2003); Maturana and Varela (1973; 1980); Eigen and Schuster (1979); Kauffman (1986; 1993)) kept defending, over the years, the inherent systemic and dynamic nature of the living, in obvious contraposition to the reductionist and rather structuralist analyses that were being implemented extensively in the fields of biochemistry and molecular biology. However, this defense was articulated through abstract models of (minimal) biological organization, very often without precise empirical correlates, targets or predictions to be later checked in the laboratory. As a result, mainstream science did not pay attention, or at least did not take any significant step in that integrative, more encompassing, systemic direction: the situation was not ripe enough yet. Things began to change considerably by the end of last century, with an apparent shift from molecular to modular (Hartwell et al., 1999) and then to systems (Kitano, 2002a,b; Westerhoff and Palsson, 2004) biology, due to a number of concurring factors. One of them was the sequencing of a good number of complete genomes, including a particularly significant one: the human genome (IHGS Consortium, 2001; Venter et al., 2001). These technological achievements led to the final realization that genomic sequences, however important, will not tell us but part of what there is to know about any biological system. Although the implementation of high-throughput experimental techniques allowed for the extraction of massive amounts of genetic data from living organisms, it was not clear how to make use of all that new information. Besides, the interpretation of those data was becoming even more complicated because, since the discovery of basic regulation mechanisms in protein synthesis (Jacob and Monod, 1961), experimental evidence had accumulated supporting the hypothesis that many regulatory modules/loops of molecules are actually operating, in a highly coordinated way, in all living organisms (see, e.g.: (Gold, 1988; Day and Tuite, 1998; Struhl, 1999)).1 Thus, it became more and more obvious to any biologist that interactions among biomolecules are at least as important as the properties of the individual biomolecules themselves. As a consequence, molecular cooperativity effects, regulation and interaction networks, synchronization processes, collective oscillatory behavior or long-range pattern formation phenomena (just to cite a few examples) turned to be the focus of attention of an increasing number 1

In particular, developmental genetics --and developmental biology at large-- had been collecting, during the previous two or three decades, an impressive body of experimental results confirming the relevance of epigenetics and regulatory networks in the formation and robustness of complex biological systems (among others, see: (Black, 2000; Smith and Valcárcel, 2000; or Graveley 2001)).

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of researchers in the natural sciences, and in cell biology in particular (Harold, 2001; Karsenti, 2008). At this stage, thanks to the earlier advances that took place especially in the field of ‘complex systems theory’ (Lewin, 1992; Waldrop, 1992; Kauffman, 1993; Solé and Goodwin, 2002), biologists had a more solid conceptual background to step on and --not less importantly-- a suite of modeling and computer simulation tools to complement and try to assimilate their rapidly growing set of empirical data. As a relevant and illustrative example, it is worth recalling that, although graph theory and network-based ideas and methods had been around for quite some time (see, e.g.: (Erdös and Renyi, 1960; Kauffman, 1969; Bondy and Murty, 1976; Hopfield, 1982)), their application to the study of biological systems experienced a very remarkable increase (maintained to date) precisely at the turn of the century, with the rediscovery (Mitzenmacher, 2003; Keller, 2005) of ‘scale-free’ and ‘small-world’ properties appearing in most of them (Jeong et al., 2000; 2001; Wagner and Fell, 2001; Strogatz, 2001; Ravasz et al., 2002). Several years later, following a different but parallel pathway, the field of ‘systems chemistry’ was launched (von Kiedrowski, 2005; Stankiewicz and Eckardt, 2006; Ludlow and Otto, 2008). Sharing several motivations and influences (it was originally proposed as a search for the connection between complex chemistries and minimal biological systems, and directly inspired by authors like Ganti (1975; 2003)),2 this field centered on the study of emergent dynamic behavior in heterogeneous molecular mixtures, typically in non-equilibrium conditions. Although its impact on the activity of chemists has not been comparable, so far, with the impact of systems biology within biology, systems chemistry has managed to bring together scientists from various areas, like supramolecular chemistry, far-from-equilibrium chemistry and prebiotic chemistry (i.e., origins-oflife-oriented research). And, in fact, it constitutes a similar shift in focus towards complex dynamic behavior, supported by a novel range of methodologies (including dynamic combinatorial chemistry, high-throughput techniques applied to populations of macromolecules, as well as microand nano-fluidics; see details below). These new methodologies have made possible, in combination with computer models and simulations, the establishment of a well-grounded scientific program to face the challenge (Ruiz-Mirazo et al., 2014). Therefore, one cannot assert that the ‘systems approach’ is new in science; and it was, indeed, the predominant standpoint in certain classical areas of biology (e.g., ecology, embryology) or chemistry (e.g., atmospheric science). However, it must be also acknowledged that the scientific transition that we are witnessing since the beginning of the 21st century has very profound 2

Tibor Ganti’s special contribution as a theoretical biologist, proposing a framework to understand the living principles grounded strictly on chemical --rather than biochemical-- stands (or, conversely, as a pioneer of ‘systems thinking’ in chemistry, driven by his deep concern and interest in biology) must be highlighted here.

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implications and does not look merely like ‘new skin for an old ceremony’: it has come to stay, and is already providing novel insights and unprecedented ways of understanding chemical and biological complexity from the very bottom, so to speak. Furthermore, we consider that the in vitro and in silico technologies recently made available (plus those in current development), if properly combined, will generate extraordinary opportunities to go much deeper in that direction, drawing increasingly accurate and comprehensive connections between the specific properties of basic material parts (molecules) and the emergent dynamic behavior of the corresponding wholes or networks (systems -- cells, in particular). This contribution is aimed to substantiate the previous claims by reflecting the state of affairs in diverse research enterprises that are pushing today’s frontiers of knowledge in order to discover the territory between chemistry and biology: a territory that remains largely underexplored and --as we will also suggest below-- can only be properly investigated through a systems lens. With that purpose, in the following section we review in more detail the historical development of the fields of systems biology and systems chemistry, as well as their main goals and methodological bases. Then, the focus will turn to those particular lines of work that are more relevant for our interests here, belonging to an area that may be called ‘prebiotic systems chemistry’. Finally, once major achievements and merits in this emerging area between chemistry and biology have been properly recognized, we will highlight several fundamental problems that remain unsolved; that is to say, key open questions that need to be tackled in order to make further progress. 2. From ‘systems biology’ to ‘systems chemistry’ 2.1. Systems Biology As mentioned above, in the last decade there has been a tremendous increase of research that bears the label of ‘systems biology’. In a search for articles that contain this term in their title or abstract, the PubMed database leads to ca. 7000 entries, out of which only three articles were reported before 2001 (Hübner et al., 2011).3 Nevertheless, a significant portion of these recent publications hit in the search use the keyword ‘systems biology’ with general classificatory purposes, referring simply to the fact that they deal with components/mechanisms of biological organisms. Thus, the demarcation of this new discipline needs to be more precisely set. Most scientists in the field conceive of systems biology as a combination of quantitative experimental data and computational modelling in order to get further insights into the dynamic behaviour of complex biomolecular networks (Camacho and Collins, 2009). This is roughly what 3

There were, of course, previous articles reporting work that nowadays would be classified as systems biology, even if the keyword was not so commonly used at that time, but they were quite marginal in any case. This PubMed search (giving the exact result of 7036 entries) was performed in early March 2015.

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O'Malley and Dupré (2005) considered as ‘pragmatic systems biology’ (a direct consequence of the ‘omics’ revolution), in contrast to ‘systems-theoretic biology’, a harder core but less numerous community of systems biologists (with stronger and older influences from theoretical biology and related fields (Boogerd et al. 2007)). Indeed, the roots of systems-level thinking in biology go far back, the actual term ‘systems biology’ being coined by Mesarović in 1968 (Mesarović, 1968). As we briefly commented in the introduction, the first developments thereafter were theoretical contributions, such as metabolic control analysis (Heinrich and Rapoport, 1974) and biochemical systems theory (Savageau, 1976), in the early 1970s, together with stochastic simulation frameworks (like Gillespie’s (1976; 1977) a bit later, which provided novel computational strategies to tackle the problem of coupled reaction systems. Subsequently, the golden age of systems biology arose from the impressive progress of molecular biology in the 1990s, with the increased ability to sequence complete genomes, analyse quantitatively molecular species and their various interactions at different levels (genome/transcriptome/proteome/metabolome), and image their spatial distribution and dynamics. The computational tools for inferring networks and building up models of their behaviour, based on those new experimental datasets, have also been growing at increasing rates. The result is a vibrant field that generates robust and high-resolution information about metabolic behaviour, regulatory networks and adaptive cellular responses (Ishii et al., 2007; Güell et al., 2009; Kühner et al., 2009; Yus et al., 2009; Nicolas et al., 2012; Buescher et al., 2012). Topics in systems biology include the study of information processing in signalling networks (Ciaccio et al., 2010; O’Shaughnessy et al., 2011), mechanistic elucidation of complex phenotypes (Goodarzi et al., 2010), as well as modelling of metabolic pathways interactions (Henry et al., 2010) and complex microbiomes (Karlsson et al., 2011). Cellular dynamics and regulation have also been thoroughly studied, with the aid of stochastic differential equations, Bayesian networks and information theory (Camacho and Collins, 2009; Hübner et al., 2011). Even if the current success of these approaches is mainly visible scientifically, some applications of systemslevel research for drug design and discovery are starting to appear, in a suggestive and likely very productive merging of systems biology with biomedical chemistry (Brown and Okuno, 2012). The simulation-experiment iterative feedback that characterizes systems biology from a methodological point of view is also favouring a shift from the discovery-driven biology of last century into a much more hypothesis-driven research paradigm (Kitano, 2002a). Our increasing capacity to understand biological systems integrally may in fact be employed to predict their behaviour under more realistic conditions, in noisy and heterogeneous environments. In this respect, a major trend of thought among modern bioengineers and systems biologists emphasizes that constructing synthetic versions of biological systems is the best approach to test the accuracy of our 6

models and hypotheses. The realisation of the potential of this approach marked the beginning of the field of ‘synthetic biology’ (Endy, 2005; Benner and Sismour, 2005; Adrianantoandro et al., 2006), with much farther-reaching implications than traditional genetic engineering (which was limited to local alterations of DNA). In brief, the ambitious goal of synthetic biology is to assemble artificial, externally designed phenotypes that show novel metabolic functions and/or genetic circuits, and in doing so provide a better understanding of cellular complexity. For instance, some remarkable advances in this field include synthetic versions of biological oscillators (Elowitz and Leibler, 2000), light sensors (Levskaya et al., 2005), genomes (Gibson et al., 2010; Baker, 2011) and chromosomes (Dymond et al., 2011; Annaluru et al., 2014). The questions and challenges posed by synthetic biology go beyond systems biology, for they involve crossing the boundaries of life as we know it today, but they are bound to be very informative for the latter, because they will surely contribute to establish the minimal requirements and organizational principles for any living system’s viability (Ruiz-Mirazo and Moreno, 2013). And, indeed, one can observe that there are already many synergies between both fields (Lanza et al., 2012). Whereas models that try to capture biological complexity in full (systems biology) will guide the design and synthesis of artificial living systems at all scales, the ability to implement or modify these systems in a controlled fashion, checking for their viability limits and other inherent constraining factors (synthetic biology) will provide a deeper knowledge of biological organisation and will also make possible the modelling of biomolecular networks with higher resolution. One of the most paradigmatic examples of the merging of ideas from these two areas, as we will discuss in more detail in section 3, is the worldwide effort to program minimal artificial cells (Solé et al., 2007; Walde, 2010). In the context of these synthetic implementations of biological or proto-biological systems, it is helpful to distinguish two types of approach. The ‘top-down’ approach pursues the assembly of components extracted from living organisms, in order to obtain constructs with modified or even improved properties than their native counterparts (Liu and Fletcher, 2009). The ‘bottom-up’ approach, in contrast, aims at assembling relatively simpler, synthetic components into a chemical system that would have the potential to become biological: i.e., a system endowed with one or several life-like properties -- such as self-maintenance, reproduction, inheritance or evolvability (Szostak et al., 2001). This second approach obviously brings the question into the field of chemistry, which already dealt in the past with the prebiotic synthesis of biomonomers and biopolymers, in close relation to the origins-of-life research. Indeed, prebiotic chemists traditionally adopted a synthetic view of this problem, characterized by the search for reactions that could lead to pure products in high yields. However, the overall failure of such a strategy has revealed the need of 7

adopting a systems perspective also in chemistry (Ruiz-Mirazo et al., 2014), since the behaviour of complex chemical mixtures is not just a simple combination of the behaviour of their molecular components (see also: (Luisi, 2006; Shapiro, 2007)). As a consequence, the field of systems chemistry has recently come to stage (Ludlow and Otto, 2008; von Kiedrowski et al., 2010), taking up the challenge to bridge chemical knowledge about molecules and supramolecular assemblies with the biological models and artificial constructs provided by systems biology and synthetic biology, respectively (see Fig. 1).

Fig. 1: Graph representing the various connections or interdependencies between those areas of chemistry and biology that are most relevant for the contents of this article. Two different levels of interaction between fields are distinguished: the coloured thin arrows link fields that are closely related to each other, either within biology (in orange) or chemistry (in red). Systems biology and synthetic biology are, for example, intimately connected and both have deep roots in the field of molecular biology. Systems chemistry, in turn, could not have emerged without the background knowledge from synthetic and supramolecular chemistry. In addition, more indirect relationships between fields, which nevertheless need to be strengthened in the future, are depicted by double-headed arrows. Generally speaking, researchers belonging to the fields on the top of the graph tend to apply more ‘systemic’ views and approaches to their scientific problems, as compared with the ones at the bottom areas, but the transition is not ‘zero to one’ (as we try to illustrate with the graded color) and exceptions to the rule probably abound.

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2.2. Systems Chemistry The term ‘systems chemistry’ was actually coined in the context of a European network meeting in Venice, in 2005, on ‘prebiotic chemistry and the origin of life’ (Stankiewicz and Eckardt, 2006), with the general aim to bring forward a new research program in chemistry that could ascertain the roots of biological complexity. Although the investigation of complex systems had been introduced and was already established in several disciplines (physics and biology included), chemists were not really taking clear steps in that direction -- with some notable exceptions: e.g., (Ganti, 1975; von Kiedrowski, 1986; von Kiedrowski et al., 1991; Bachmann et al., 1992; Lee et al., 1996; 1997; Lehn, 1999; Cousis et al., 2000). This was in sheer contrast with the generally acknowledged fact (Luisi, 2006) that many chemical mixtures generate emergent properties -- i.e., properties that cannot be attributed to any of the mixture components, acting in isolation, but result from the interactions among all components. Thus, the underlying intuition was that the key for finding thermodynamically feasible routes towards the first living organisms lied in the study of more dynamic (i.e., less robust (Eschenmoser, 2007)) and diverse reaction cycles (e.g., coupled autocatalytic cycles a la Ganti (Szathmáry et al., 2005) -- see below), making use of prebiotically relevant compounds, but combined in higher levels of molecular heterogeneity than so far tried (Szostak, 2011). The general objective of systems chemistry to investigate mixtures of multiple chemicals and their dynamic interactions was articulated through insights coming from two main different fields (von Kiedrowski et al., 2010). First, the influence of systems biology must be acknowledged, for many tools and ideas that systems chemists are currently employing have been inherited from that field, as sketched above. Nevertheless, the great advances of supramolecular chemistry during the last 40 years, providing an ever wider set of self-assembling and self-organizing materials, also played a critical role, because this brought about a new generation of chemists with the skills and the courage for trying to come to terms with networks and assemblies (Stoddart, 2012). In that context, the appearance of dynamic combinatorial chemistry (DCC) at the beginning of the present century (Lehn, 1999; Cousins et al., 2000; Lehn and Eliseev, 2001) represents a good indicator of the beginning of such a transition, from purely synthetic chemistry into a systems perspective. In words of von Kiedrowski and colleagues (2010), systems chemistry would seek to combine the ‘classical’ knowledge of synthetic and supramolecular chemistry with the ‘classical’ knowledge derived from extant forms of life. More specific goals of the field include the evaluation of structural and dynamic requisites leading to molecular self-replication (Kindermann et al., 2005), the study of chiral symmetry breaking (Kuhn, 2008; Blackmond, 2011), the integration of various (bio)chemical processes within 9

protocellular structures (Luisi et al., 2006; Rasmussen et al., 2008; Chen and Walde, 2010), and the quest for the roots of Darwinian evolution in chemical systems (Szathmáry, 2006; Pross, 2009). In any case, given the bottom-up, synthetic character of the systems chemistry approach, these tasks do not necessarily have to be performed with biomolecular building blocks. So systems chemistry has, in this sense, a much wider scope than systems biology, because it also asks itself whether complex, proto-biological systems could be constructed from artificial components alternative to those chosen by nature more than 3.5 Ga ago. Accordingly, research on synthetic nucleic acid analogues, metabolic networks based on novel (bio)chemistries, and fatty acid --or even polymer-compartments instead of phospholipid ones, are considered as worth-pursuing approaches: they allow exploring complex, emergent properties in chemistry without the restrictions imposed by the historical pathway that biological evolution on Earth actually followed (Ruiz-Mirazo et al., 2014). From a methodological point of view, the success in the merging of systems biology and synthetic/supramolecular chemistry into systems chemistry would have been impossible without the recent, impressive developments of nanotechnology and analytical chemistry. Thus, chemists have also contributed to the shift by abandoning, in the last decades, the ‘security zones’ of solutionphase and solid-state chemistry, addressing processes that take place in soft matter (Minkenberg et al., 2009), on surfaces (Orentas et al., 2012) and at interfaces (Belenguer et al., 2011). Within this new paradigm, there is also an obvious need to spatially control the organization of molecules in one-, two- and three-dimensional space, so their properties and functions coherently emerge over the whole range of sizes, from nano- to macroscopic architectures (Whitesides and Gryzbowski, 2002). The target here is producing robust hybrid natural/artificial materials, preferably compartmentalized, to improve our manipulation/control capacities over them. On the analytical side, in turn, it has become possible to characterize moderately complex mixtures of molecules without the need of isolating individual components: this is typically done through a combination of methods, including high- or ultra-performance liquid chromatography (UPLC) coupled to mass spectrometry (MS) and multidimensional nuclear magnetic resonance (NMR) (Nitschke, 2009). Techniques coming from systems biology and high-throughput biotechnology are also being very useful in this respect: namely, DNA (Shendure and Lieberman Aiden, 2012) and peptide (Shively, 2000) sequencing, microarrays and biochips (Marks et al., 2007), together with microfluidics (Atencia and Beebe, 2005). In addition to these common tools, the methods that have contributed more specifically to the development of systems chemistry are those involved in DCC, defined as combinatorial chemistry under thermodynamic control (Corbbet et al., 2006; Li et al., 2013). In principle a dynamic combinatorial library (DCL) is formed by a set of molecules that are inter-convertible 10

through reversible covalent or non-covalent bond formation processes (for a review on DCLs and their multiple applications, see (Li et al., 2013)). Nevertheless, an exciting new branch of DCC is exploring the combination of equilibrium steps with kinetically controlled processes such as catalysis, autocatalysis and self-replication (Giuseppone, 2012). The resulting far-from-equilibrium systems open unprecedented opportunities to explore analogies with biological phenomena (Biosa et al., 2006). For instance, dissipative structures formed by self-assembly of some network components have shown ‘functions’, like directional movement (von Delius et al., 2010) and adaptive self-replication (Nguyen et al., 2009; Carnall et al., 2010). These achievements support farfrom-equilibrium systems chemistry as one of the most promising approaches on the way towards synthetic protocells, if properly integrated with insights and developments from the supramolecular chemistry field. That integration will require, in any case, the combination of experimental results with computational studies, a further methodological similarity between systems chemistry and systems biology. Even if simulation platforms and theoretical models to deal with chemical complexity in silico are still behind those developed for biology (for a couple of exceptions, see: (Dittrich and di Fenizio, 2009; Mavelli and Ruiz-Mirazo, 2010)), they are fundamental to provide a coherent picture and interpretation of in vitro results -- or, even, go beyond them. As an illustrative example, the simulation of a set of DCLs with a varying number of chemical compounds (from 65 to 4.828) confirmed that the use of larger libraries is advantageous in terms of obtaining --more rapidly and reliably-- certain final properties in the emerging collection of molecules (e.g., improved and selective binding affinities) (Ludlow and Otto, 2010). Theoretical modelling work and computer simulations are especially valuable because they allow us to analyse in much deeper and exhaustive ways the dynamics of these complex, multi-variable systems, which is one of the main challenges that this field has ahead, as we will highlight in the following sections. In particular, we will focus on those areas and lines of research that are specifically addressing the connection between the chemical and biological domains: i.e., scientific enterprises that are currently exploring various hypothetical pathways to bridge the --still enormous-- gap between the inert and the living worlds.

3. ‘Prebiotic systems chemistry’: main achievements and challenges The route from the first biologically relevant chemical processes and molecular building blocks to the first full-fledged living organisms required the transition from inorganic to organic chemistry, the development of functional biopolymers and, together with it, the progressive coupling of genetic mechanisms, membrane-based compartments and protometabolic cycles to generate self11

reproducing and self-sustained cellular systems. Miller and Urey’s experiment in 1953 (Miller, 1953) exported the problem of the origin of life from the field of theoretical biology to the experimental sciences, thus inaugurating the age of prebiotic chemistry. Since then, different synthetic approaches have shown that the abiotic production of amino acids and other biomonomers in significant amounts could have been achieved in allegedly early Earth conditions, provided that mixtures of reactive molecules were exposed to adequate energy sources and catalysts. Some of the building blocks for biological molecules could have been synthesized, alternatively, in the interstellar medium and delivered to Earth through extraterrestrial bodies, including meteorites and cometary nuclei. It is currently assumed that, as a result of the combination of endogenous and exogenous chemical processes, a rich repertoire of compounds might have been produced abiotically. In fact, experimental support in favour of this hypothesis has accumulated, over the last six decades, thanks to numerous research programs that have investigated plausible chemical pathways toward the main families of biomonomers: lipids (or simpler membrane-forming amphiphiles), amino acids, sugars, and nucleotides -- reviewed in (Ruiz-Mirazo et al., 2014). Despite the significant advances that have been made in the prebiotic chemistry field, subsequent critical steps, like the advent of template replication, the emergence of autocatalytic reaction networks or the coupling between proto-genome replication and compartment reproduction, are still far from being understood (Szathmáry and Maynard-Smith, 1997; Shapiro, 2007; Eschenmoser, 2007; Szostak, 2012). These are key transitions for the emergence of the first living organisms, since such systems must combine (Ganti, 2003) the ability to: i) distinguish themselves from their environment and keep their molecular components together, thanks to a selectively permeable boundary; ii) stay away from thermodynamic equilibrium by gathering energy and material resources from the environment, thanks to the action of metabolic cycles; and iii) transmit heritable information to their progeny, thanks to the presence of genetic molecules and mechanisms. Noticeably, the physicochemical mechanisms suggested to have been involved in the formation of infra-biological subsystems (i.e., membrane compartments, metabolic machineries and genetic mechanisms, separately considered) often appear as incompatible (Szathmáry et al., 2005). If this was the case, the necessary integration of those subsystems would become a utopia. As an alternative and more realistic scenario for the origins of life, a systems chemistry based approach is now being put forward. On these lines, it has been recently demonstrated, for instance, that various precursors of ribonucleotides, amino acids and lipids could all derive from the chemistry of hydrogen cyanide, occurring in rather elaborate but prebiotically plausible geophysical conditions 12

(Patel et al., 2015). More generally speaking, such a ‘prebiotic systems chemistry’ view (RuizMirazo et al., 2014) would be based on the assumption that heterogeneous aqueous solutions of different monomers and oligomers coexisted in the pre-cellular world, and that different catalytic species might have been present, including metals, mineral surfaces, and reactive interfaces with water-based media. Thus, it seems that the traditional gene-first vs. metabolism-first controversy will be progressively substituted by a scenario in which all the basic molecules co-evolved from the beginning in different (though sooner than later interconnected) environments, forming heterogeneous, pre-biochemical interaction networks. This more integrative approach, all the way from the very beginning, could help to explain the transition from complex (but still just thermodynamically driven) chemical systems into proto-biological ones and, eventually, into mature living organisms (where kinetic and spatial control of reactions take over -- see a more detailed discussion below, in section 4). Even if some of the results obtained following this new and wider conception (Powner et al., 2009; Patel et al., 2015) make us hopeful, the experimental implementation of complex reaction systems is a remarkably difficult task, since a number of --not necessarily compatible-- chemical processes must be performed synchronously, or in a defined set of subsequent steps, and involving diverse mixtures of molecular species and catalysts. A possible way to tackle the problem, making it more amenable, is to modularize it -- i.e., decompose it into relatively simplified ‘sub-problems’. For instance, inspired by Ganti’s scheme of the chemoton (Ganti, 1975; 2003), we could reorganize our approach subdividing the target system (i.e., the biological system) into three hypothetical subsystems, each of which would be, in itself, a chemical supersystem. According to the chemoton theory, a minimal living cell would consist of a metabolism (represented by an internal autocatalytic reaction network), a self-replicating polymer (or set of polymers) carrying genetic information, and a globally encapsulating membrane (whose molecular constituents are produced by the metabolism), all stoichiometrically coupled. This general framework was, in fact, further elaborated to that aim by Szathmáry and colleagues (2005) and can be used to illuminate new research pathways or classify those in current development within the field of systems chemistry. Thus, by connecting the protomembrane, protometabolism and protogenome subsystems into doublet (‘super-chemical’ though still ‘infra-biological’) systems, key intermediate stages in the origins of life process can be more clearly established, helping to identify major landmarks and articulate better-focused investigation avenues towards the bottom-up construction of the ternary supersystem that should fulfil all the requirements for life.

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Indeed, a wide variety of experimental systems developed over the last decade, either from a synthetic biology or origins of life research perspective, can be adequately interpreted in terms of those three binary combinations. For instance, the implementation of an autocatalytic reaction network of prebiotic interest, like the sugar-synthesizing formose reaction, within liposomes (Gardner et al., 2009) represents an obvious example of a ‘membrane-metabolism’ binary construct. Similarly, the lipid encapsulation of different catalytically active minerals (including FeS, CdS and TiO2) (Vassiltsova et al., 1999; Summers et al., 2009; Alpermann et al., 2011) to assess the energy harvesting potential of a protocellular system (as a prerequisite for the synthesis of complex biomolecules) should be included in the same category. Other relevant achievements of this ‘compartmentalized-chemistry’ field include the nonenzymatic polymerization of peptides (Hitz et al., 2001; Zepik et al., 2007) and nucleic acids (Rajamani et al., 2008; Olasagasti et al., 2011) in liposomes and multilayered lipid phases. More recently, working also towards the integration of protometabolism and boundary, the group of Szostak reported the synthesis of the dipeptide AcPheLeuNH2 by the catalytic dipeptide Ser-His encapsulated in oleic acid vesicles. The synthesized peptide binds to vesicle membranes, thus increasing their affinity for fatty acids and promoting vesicle growth (Adamala and Szostak, 2013a). This suggests that adaptive changes and competition could have initiated in peptide-containing protocell vesicles. In turn, the experimental efforts towards the integration of genetic molecules and metabolism have been based on the investigation of the catalytic properties of RNA molecules (natural and in vitro-evolved ribozymes) and ribonucleoprotein complexes (Puerta-Fernandez et al., 2003; Lilley, 2005; Cech, 2009), which rely on the ability of RNA to interact with different kind of ligands (Kondo and Westhof, 2010). Indeed, the binding of small cofactors to RNA molecules might have broadened the catalytic repertoire of ribozymes during the RNA world, as exemplified by the reported coenzymatic use, in current organisms, of a monosaccharide (glucosamine 6phosphate) as part of the glmS ribozyme-riboswitch (Ferre-D’Amare, 2011). The characterization of different RNA-binding peptides suggests that peptidic cofactors could have also operated in prebiotic times (Frankel, 2000), probably fine-tuning the evolution of catalytic function from ribozymes to present day enzymes (Cech, 2009). Although a still limited number of experimental approaches have been devoted to in vitro evolve RNA molecules in the presence of peptides, some functional ribonucleopeptide complexes have been obtained, including those that bind ATP (Hagihara et al., 2004; Nakano et al., 2011) or phosphotyrosine (Nakano et al., 2008). Based on these successful examples, the generalization of in vitro experiments aimed at evolving RNA molecules (endowed with both genetic and catalytic functions) in the presence of prebiotically 14

relevant mixtures of molecules (such as amino acids and peptides, sugars, antibiotics or other kinds of low molecular weight compounds) should represent a further extension of the systems-based combination of template and metabolism. The third binary system, consisting in the integration of compartment and template molecules, has been approached by the encapsulation of genetic polymers within self-reproducing vesicles formed by prebiotically plausible amphiphilic molecules. Pioneering experiments showed that nucleic acid polymerization reactions could proceed in the lumen of vesicles provided that the required protein enzymes are also encapsulated (Walde et al., 1994; Chakrabarti et al., 1994; Oberholzer et al., 1995; Shohda and Sugawara, 2006), although the effective coupling between template replication and compartment growth or division was not achieved. Other research programs have investigated the compatibility between nucleic acid molecules (RNA, DNA, or nucleic acid analogues -- XNAs) with phospholipid vesicles, addressing relevant questions that include the permeability of the membrane upon binding of RNA molecules (Vlassov et al., 2001) and the possible role of RNAs as early membrane transporters (Janas et al., 2004). The group of Szostak has shown that fatty acid vesicles containing osmotically active molecules, in particular RNA oligomers, could grow and potentially divide more quickly, thus leading to the emergence of competition in RNA-boundary protocell populations (Chen et al., 2004). This group has progressively advanced, as well, in the nonenzymatic template-directed polymerization of RNA inside vesicles (reviewed in: Schrum et al., 2010; Szostak, 2012), including a more recent report showing that the presence of citrate inside the compartment protects fatty acid membranes from the disruptive effects of the high Mg2+ concentrations required for RNA copying (Adamala and Szostak, 2013b). However, all these experimental milestones still lack a genome-driven coupling between the replication of the template molecule and the reproduction of the compartment itself (Zepik and Walde, 2008). As theoretically schematized by the main players of this research program more than a decade ago (Szostak et al., 2001), that genome-compartment connection would require the endogenous, ribozyme-catalyzed synthesis of one amphiphilic molecule from its precursor substrates, the amphiphile being then incorporated into the membrane at a rate proportional to its abundance in the vesicle lumen. Such a (still unknown) ribozyme should have been copied by an (also hypothetical) RNA replicase ribozyme able to polymerize --through a template-directed mechanism-- the two RNA sequences that would constitute the ‘bipartite genome’ of the protocellular system (see also: Mavelli, 2012). However, even in this case, the autonomous maintenance of the ‘ribocyte’ (i.e, the RNA-based, genome-compartment binary system, as

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envisioned) would necessarily involve a number of additional metabolic reactions taking place within the vesicle. In parallel to the experimental approaches towards the assembly of doublet, infra-biological systems, a number of research lines have focused on the direct synthesis of ternary systems: i.e., minimal but supposedly complete living beings. Their goal is the construction of an artificial cell, formed by a lipid membrane encapsulating an informational polymer able to replicate itself, the entity as a whole being endowed with the proper metabolic components and processes that allow the exchange of matter (nutrients/waste products) and energy with its environment. This ambitious enterprise has become feasible thanks to the merging of liposome and cell-free-extract research technologies (reviewed in: Stano et al., 2011), a combination that has led to diverse semi-synthetic (rather than bottom-up or strictly artificial) ‘bioreactors’ (Luisi et al., 2006). In a particularly interesting case (Noireaux and Libchaber, 2004), a plasmidic DNA molecule was entrapped in lipid vesicles, together with an E. coli extract that contained the transcription and translation machinery required for the expression of two encoded functionalities: a pore-forming protein that ensured nutrient accessibility by increasing membrane permeability, and a fluorescent protein used as a marker for protein synthesis. This bioreactor produced proteins for up to 4 days and the system was further refined to avoid the use of total bacterial extracts (Chalmeau et al., 2011). Temporary metabolic activity was also achieved in similar semi-synthetic cellular constructs, but employing a collection of selected recombinant proteins (e.g., the so-called ‘PURE system’ (Shimizu et al., 2001)). Making use of an alternative approach, Yomo’s group (who were the first to carry out the biosynthesis of several well-folded proteins within lipid compartments (Ishikawa et al., 2004)) reported RNA replication by a self-encoded RNA polymerase enzyme in liposomes that contained a reconstituted reaction system composed of 144 gene products, including an in vitro translation system (Kita et al., 2008). Despite the biotechnological relevance of these accomplishments and their key role as ‘experimental proofs of principle’ (in particular for areas like synthetic biology), current research avenues in bioreactor science do not really come to terms with ‘bottom-up’ construction principles: namely, they provide no clues about a hypothetical sequence of steps of increasing complexity (like the one schematically represented in Fig. 2), so they do not illuminate any feasible pathway for the origins of life. Relatively simpler schemes have been tried (e.g., the combination of peptide nucleic acids as genetic molecules and a light sensitizer in a lipid compartment, which showed potential for growth and division driven by the photocatalytic production of its membrane components (Rasmussen et al., 2004; DeClue et al., 2009)) but difficulties for full system integration are 16

apparent and the prebiotic plausibility of some of the molecular ingredients involved is null. Indeed, as we will discuss in more detail in the next section, several major issues regarding these protocellular constructs remain open, like the effective coupling between various subsystems (e.g., the replication of the genetic molecule and the growth/reproduction of the compartment, as mentioned above), or the reliable reproduction of the system as a whole, ensuring the proper distribution of molecular constituents among daughter protocells (which would truly constitute the substrate of Darwinian evolution). Some experimental approaches are addressing more directly these problems, like a recent work connecting membrane dynamics and metabolism (namely, encapsulated enzymecatalized reactions that are activated or inhibited by the addition of charged amphiphiles upon vesicle fusion (Caschera et al., 2011)) or the report on growth and division of vesicles triggered by the amplification of an enclosed DNA molecule (through a process leading to roughly equivalent distributions of the DNA templates among daughter protocells (Kurihara et al. 2011)). However, the challenges ahead in this area are many and diverse, and require the concerted effort of a bigger number of researchers pursuing systemic integration strategies.

Fig. 2: The systems chemistry perspective applied to draw a hypothetical bottom-up sequence of prebiotic transitions leading to living cells, including the main molecular ingredients involved. See text for details. 17

4. Perspective: some open problems and tentative ideas As already pointed out above, biological systems consist of very diverse but --at the same time-highly interdependent molecular components. Unless we find alternative and simpler examples of life elsewhere (on a different planet/satellite, or in a synthetic biology lab), this fact could be revealing that the robustness of living organisms --and the living phenomenon in general-- is based on the capacity of these systems to achieve the tight functional integration of different chemical species. By functional integration here we mean that some chemical species contribute to the synthesis --or maintenance-- of some others which, in turn, contribute to the synthesis --or maintenance-- of some others…, until the whole set (i.e., what comes to constitute the system) achieves a dynamic state of relative stability (a self-maintaining stationary state, which also includes the possibility for growth and reproduction -- see Fig. 2). As many authors have pointed out before us (see, for instance, (Morowitz, 1968; Ganti 1975; Prigogine, 1980) or, more recently, (Harold 2001; Eschenmoser, 2007; Pross, 2009; Pascal et al., 2013)) the chemical bases for this integrative and dynamic stability on which biological organization thrives cannot be found in or around equilibrium conditions (i.e., through the minimization of an energy function). Most biomolecules are actually transient, involved in a continuous process of cyclic transformation (metabolic turnover) that takes place within the cell, in out-of-equilibrium conditions, thanks to the adequate inflow and outflow of material and energetic resources; and thanks, as well, to the presence of other biomolecules (the genes or, rather, the whole cell DNA, the genome) that stay there, practically unaltered --in characteristic metabolic time scales-- as a reference for the rest, so that the system does not lose its complexity and eventually decays (von Neumann, 1966). Scientific evidence from different fields confirms that these ‘genetically instructed cellular metabolisms’, built upon a common essential biochemistry, have been around for at least a few thousand million years, and have therefore demonstrated their capacity (collectively speaking) for propagation, adaptation and long-term sustenance on a planet like the Earth. Obviously, given the astonishing level of molecular and organizational complexity involved in any genetically instructed cellular metabolism that we know (even in the simplest bacteria, like Mycoplasma), a scientific approach to the question of life’s origin must necessarily assume intermediate stages, in-between chemistry and biology, with intermediate levels of stability or robustness of the entities involved, and construct, ‘from the bottom-up’, a plausible sequence of transitions connecting those diverse stages. Experimental research at the crossroads between 18

chemistry and biology is a very hard task, though, especially because we lack natural examples -and, so far, also the necessary artificial or synthetic ones-- to illustrate what those stages might be like. Nevertheless, one can safely assume that the type of chemical stability to be investigated should be inherently different from the one associated to systems under ‘thermodynamic control’, which typically lead to equilibrium or quasi-equilibrium molecular structures -- even if some of these structures could also play biologically interesting roles. Accordingly, more attention ought to be turned to the development of cyclic networks of chemical reactions where various forms of ‘kinetic control’ could be realized among different components, keeping the majority of species away from thermodynamic equilibrium, as previously suggested (Eschenmosser, 2007; Pross, 2009; 2011). However, acknowledging the importance of this kinetic aspect (also supported by the central role played by enzymes in any metabolism), we consider that a more radical move is required – i.e., not simply a shift from thermodynamic to kinetic stability perspectives. In order for a chemical system to achieve dynamic stability through the functional integration among different parts --and start becoming, therefore, of biological significance-several aspects should be taken concurrently into account, right from the beginning, and all the way through, until the end of the process of origins. One of them is, naturally, the temporal coordination of the diverse chemical reactions involved, and the development of catalysis (i.e., kinetic control mechanisms) is fundamental in that sense, as we just commented above. Insights about the hypothetical transition from mineral catalysis and organocatalysis towards (RNA or protein-based) enzyme catalysis would be of high relevance on this front, together with work on the spontaneous emergence of temporal patterns of behavior, synchronization phenomena, etc., within nonequilibrium chemistry. Nevertheless, other basic organizational problems are bound to require some sort of solution from the start. In particular, the problem of molecular diffusion and the general tendency towards spatial homogenization is also a central one. The question under focus here would be how to generate chemical microenvironments that distinguish themselves from the surrounding milieu and have, therefore, chances to become progressively more complex than the latter. On these lines, although ‘two-dimensional’ reaction dynamics could be distinctively established on diverse regions of --both inorganic and organic-- surfaces, control on the concentrations and spatial flux of materials by means of their encapsulation within self-assembling membranes with selective permeability is bound to take over soon. If that is the case, research on protocellular systems becomes the most reasonable and experimentally tractable way to tackle this aspect. Furthermore, the combination of aqueous and lipidic domains in the same setting implies the presence of heterogeneities and soft organic interfaces that can also be helpful to overcome another difficulty 19

which is seldom addressed in prebiotic investigations: the thermodynamic impossibility or endergonicity of many biochemical reactions. In particular, polymerization reactions (both for the synthesis of oligonucleotides and oligopeptides) always entail the release of water molecules and, therefore, are not spontaneous in aqueous solution. This is a very severe problem, whose solution goes surely beyond the possible contribution of hydrophobic domains and interfaces: it requires, as well, the acquisition and efficient management of energy resources across the system (i.e., transduction mechanisms) and the use of intermediary compounds (equivalent to present day energy currencies, like ATP) to couple endergonic and exergonic processes of various kinds (Ruiz-Mirazo and Moreno, 2004). Last but not least, in order to avoid an eventual decay and have the possibility to increase in complexity, these systems need to ‘fix’ or ‘register’ molecularly their fundamental features and be able to propagate them reliably (von Neumann, 1966). Template mechanisms (like the ones articulated on polynucleotide chemistry) are the ones expected to ‘come to rescue’ in this regard. Figure 2 summarizes, very schematically, the type of ingredients that we are referring to, and their functional engagement in a protocellular entity with potential for growth and division. One could open the discussion about other aspects or components that might also deserve to be included in this initial set but, in any case, the key point to be highlighted here is that, even for the earliest steps of prebiotic chemistry, a diverse combination of molecular ingredients and processes might well be required. The intuition behind this conceptual change is that a certain number of different ‘chemical tasks’ (not just catalysis, but also transduction mechanisms, spatial confinement, mediated diffusion or template activity) may need to be jointly performed in order to ensure a minimal level of dynamic stability or robustness, even in the simplest infrabiological systems. So, what we are suggesting is that, apart from considering single/doublet autocatalytic ‘super-chemical systems’ or other ‘proto-chemoton’ alternatives (Griesemer and Szathmáry, 2009) as more plausible initial steps, one should also directly try, for starters, ‘triplet’ or ‘quadruplet’ ones. In other words, making use of much simpler molecular species than current biopolymers --like de Duve (1991) and Shapiro (2006; 2007) already suggested, among others--, one should try to implement relatively complex schemes in terms of component diversity and variety of chemical roles that they could play. If this hypothesis of a necessarily heterogeneous scenario is found correct,4 we will understand why classical prebiotic approaches focusing on synthetic shortcuts to just a particular type of biomolecule (peptides, nucleotides, lipids or sugars) have not been that successful. Besides, traditional controversies about the primordial scenario for origins (‘replication-

4 Note that our claim for heterogeneity goes quite far beyond other, more cautious suggestions on similar lines, like Szostak’s (2011).

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first’ and ‘metabolism-first’ schools), based on favoring single biopolymer types (typically nucleic acids vs. proteins), will progressively fade away. In other words, the systems approach would be confirmed as indispensable along the whole process of origins of life, not just when the main final molecular players (i.e., phospholipid compartments, RNA, proteins, DNA) appear on the scene and ‘suddenly realize the benefits of staying together’. From this perspective, integration difficulties should be faced upfront (as Szostak (2012) proposes for the compartmentalization problem), simply because leaving them for later makes things still worse. Most of the trouble that experimental researchers are finding in their attempts to combine complex biomolecules within a protocell (see previous section) has to do with the functional coupling of the different species they work with. So perhaps what needs to be changed is the general approach to the problem (like Sutherland did for the specific case of the prebiotic synthesis of nucleotides (Powner et al., 2009)), bringing the issue of integration all the way back to the very beginnings. On these lines, the crucial question would be to determine experimentally the initial conditions, the heterogeneous set of elementary compounds and processes that would spontaneously generate a population of chemical systems showing an enhanced dynamic stability in a nonequilibrium situation. When the number of variables and parameters of control increases, like in this proposed scenario, the space of possibilities for exploration also increases, but in an exponential way. Therefore, some criteria are required, in practice, to tame such a combinatorial explosion. An obvious one is the prebiotic plausibility of the starting materials and geophysical/geochemical conditions. Then, a no less important point is the principle of continuity with biological systems, as we know them on Earth. For instance, the option that we are illustrating in Fig. 2 would entail the functional engagement of four main types of ingredients (membranes, catalysts, energy currencies and templates) that become increasingly complex and interdependent as protocells develop and reach higher levels of robustness in their dynamic behavior. This is because we are making an ‘informed guess’, projecting from extant biology and, more specifically, from the fundamental functions that different biomolecules efficiently carry out in all living cells, after a long process of evolution and optimization through natural selection. But, regardless of the particular combination of ingredients/processes to be tried, the overall idea would be to think about a collection of precursor molecules that could be fulfilling similar roles to the currently performed ones. Surely less efficiently, but already coupled together; i.e., being part of a more precarious --yet, relatively autonomous-- system.

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Another idea that lies somehow implicit in Fig. 2 and could be quite relevant --at least worth highlighting in this context-- regards a transition that should take place towards systems that become increasingly capable of synthesizing their own functional components. In the first stages of the process of protocell development, as depicted in the figure, the most reasonable assumption is that vesicles make direct use of available resources in the environment, which may access the compartment by passive diffusion. However, the cohesion and dynamic robustness (and, thereby, the adaptive capacities) of the protocellular system are bound to increase significantly when it begins to synthesize its own functional machinery. This occurs, for instance, when energy-rich compounds or organic/metal/mineral catalysts initially found in the surrounding milieu start being substituted by endogenously produced energy currencies or catalytic molecules that outcompete the former in efficacy; or when membranes change their composition and biophysical properties as a result of the uptake of new, internally synthesized lipid molecules (Ruiz-Mirazo et al., 2011; Budin and Szostak, 2011). In that sense, the capacity for ‘self-production’, even if this is never achieved completely (the system, at any rate, must remain thermodynamically open), could be of central importance in the process of enhancing functional diversification, integration and robustness (i.e., plasticity). How soon in protocell evolution should this property come to stage is an open issue, but probably deserves closer empirical examination. The evolutionary capacities and limitations of these hypothetical protocellular systems, as such, would also be an important subject for exploration. Experiments carried out in Szostak’s lab during the last decade (Chen et al., 2004; Budin and Szostak, 2011) have demonstrated that relatively simple vesicle populations can get involved in competition dynamics for the lipids available in the environment and, depending on various physicochemical factors (like the osmotic state or the lipid composition of the membrane), show different growth rates. However, the problem of vesicle reproduction, despite some reported progress (Takakura et al., 2003; Stano et al., 2006; Zhu and Szostak, 2009), remains elusive. Biological research on division mechanisms is showing that bacterial fission could be induced simply by excess lipid production (Mercier et al., 2013; Errington, 2013). But the difficulties stem from the lack of internal mechanisms, at these early stages, to promote regular patterns of variation in the global shape of the protocell that could bring it closer to a division event, plus the necessary energy input for the final --remarkably uphill-membrane fission steps. Therefore, although stochastic reproduction is always a possibility, stationary growth-division cycles of this type of systems are not easy to achieve, and the task becomes still more complicated if their overall molecular composition is expected to be maintained across generations (Mavelli and Ruiz-Mirazo, 2013). 22

One of the keys to overcome these difficulties, again, may be found in the idea of functional coupling. Regular reproductive cycles with the possibility to avoid progressive size decrease or dilution effects must be based on a growth process fundamentally driven by internal chemical syntheses: the volume of the system will only tend to grow steadily provided that there is a net increase of internal compounds (e.g., through endogenous autocatalytic loops). In other words, the protocell must be a --somehow controlled-- factory of molecules, including its own boundary components (membrane surface has to grow harmoniously, too), which would be an additional way of fostering division. Even if this ‘molecular factory’ or ‘proto-bioreactor’ was rather modest initially (compare Fig. 2 with the level of complexity of any current metabolism), it should produce all molecules that the system needs for its stability and are not readily available in the environment: otherwise, multiplication events will sooner or later dilute away any system ingredient (Mavelli and Ruiz-Mirazo, 2013). Concomitantly, genetic mechanisms to fix and reliably transmit important molecular innovations (e.g., the appearance of a more effective catalyst for a reaction, or a membrane component that enhances the permeability to a given nutrient) would be required, given the constant process of competition that would already be taking place among protocells in any shared environment. Then, the genetically-controlled production of amphiphilic membrane components by an encoded catalyst could have efficiently coupled (proto)genome replication and protocell reproduction, a requisite for the genotype/phenotype dichotomy that constitutes the substrate of biological evolution. Therefore, in a similar way as it should happen with the degree of functional integration and dynamic robustness of these prebiotic systems, their evolutionary potential would also be expected to increase across those successive infrabiological stages. In this context, the production of molecules with both template and catalytic properties (e.g., short polynucleotides similar to current RNA, likely following stepwise processes of structural and functional complexification (Briones et al., 2009)) is bound to have radical effects both in terms of protometabolic efficiency/robustness and evolutionary dynamics. This is, in fact, one of the main ideas guiding the origins of life research for the last ten years or so: the quest for the ‘ribocell’ (Szostak et al., 2001; Mansy et al., 2008; Adamala and Szostak, 2013b). Indeed, if an adequate coupling between chemical reaction processes, membrane dynamics and reliable enough hereditary mechanisms (i.e., copying mechanisms that conserve molecular sequences but allow for variation, and inheritance of this variation) is experimentally achieved, this will certainly constitute a major breakthrough in the field. It would in practice represent the transition from pre-Darwinian modes of competition and selection (in growing and --perhaps reliably-- multiplying protocell populations), towards an evolutionary process that gets much closer to biological open-endedness. The ‘unlimited 23

heredity’ potential that long enough oligonucleotides of the protogenome would bring about (Maynard Smith and Szathmáry 1995) would be crucial in that sense, but provided that they are functionally integrated in more complex and compositionally diverse protocellular entities resembling chemotons (Ganti 1975).

5. Concluding remarks Exploring the territory between chemistry and biology, and trying to explain how the transition from the former to the latter could have occurred, at least once in this minute region of the universe that we inhabit, requires looking into many different physicochemical aspects and suggesting plausible mechanisms for the functional integration of different chemical species in complex, heterogeneous molecular mixtures. Thus, the necessity to adopt a systems perspective in the chemical sciences has turned evident. While this systems view had already penetrated in various disciplines, including physics and biology, its settlement in chemistry is relatively new, although extremely promising. The main goal of systems chemistry is to ascertain the roots of biological complexity, with the aid of both traditional and recently developed methodologies, including those employed in more reductionist approaches like organic synthesis and supramolecular chemistry. In contrast to synthetic biology (whose means to build up minimal artificial protocells can perfectly involve components extracted from extant living organisms), the research programs that explore prebiotically plausible pathways to assemble proto-living chemical systems (i.e., compartmented systems showing dynamic self-maintenance, autonomous behavior, reproduction, inheritance and evolvability) constitute what we have termed ‘prebiotic systems chemistry’. This task is really challenging from an experimental point of view, especially with regard to the coupling of genetic mechanisms, protometabolic cycles and membrane-based compartments. In any case, we consider that an integrative systems strategy should be tried from the very beginning of the whole process of biogenesis, in agreement with previous proposals that envisioned a rather ‘dirty’ and heterogeneous chemistry for the early origins of life (Dyson, 1985; de Duve, 2007). In this area of research, like in many others, the transition to a broader systemic view has become inescapable; the advantage here is the astonishing amount of detailed knowledge that science has provided, over the years, on the behavior of molecules -- biomolecules in particular. Let us make use of all that analytic knowledge in fruitful, synthetic ways.

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Acknowledgements AdlE, Ramón y Cajal Research Fellow, received support from the Spanish MICINN (CTQ201124187/BQU) and the Comunidad de Madrid (MADRISOLAR-2, S2009/PPQ/1533). The work of CB was supported by the Spanish Ministry of Science and Innovation (MICINN Grant BIO201020696) and Spanish Ministry of Science (MINECO Grant BIO2013-47228-R). KRM acknowledges support from the Basque Government (Grant IT 590-13), MINECO (Grant FFI2011-25665) and COST Action CM 1304 (‘Emergence and Evolution of Complex Chemical Systems’). CB and KRM also thank the interdisciplinary framework provided by COST Action TD 1308 (‘Origins and evolution of life on Earth and in the Universe’). Finally, KRM would like to thank Eörs Szathmáry for the invitation and excellent organization of the Badacsony Systems Chemistry Meeting, in memory of Tibor Gánti, during which many of the ideas included in this paper were presented.

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