Abstract It is very likely that life began with some RNA (or RNA-like) molecules, self-replicating by base-pairing and exhibiting enzyme-like functions that favored the self-replication. Different functional molecules may have emerged by favoring their own self-replication at different aspects. Then, a direct route towards complexity/efficiency may have been through the coexistence/cooperation of these molecules. However, the likelihood of this route remains quite unclear, especially because the molecules would be competing for limited common resources. By computer simulation using a Monte-Carlo model (with “micro-resolution” at the level of nucleotides and membrane components), we show that the coexistence/cooperation of these molecules can occur naturally, both in a naked form and in a protocell form. The results of the computer simulation also lead to quite a few deductions concerning the environment and history in the scenario. First, a naked stage (with functional molecules catalyzing template-replication and metabolism) may have occurred early in evolution but required high concentration and limited dispersal of the system (e.g., on some mineral surface); the emergence of protocells enabled a “habitat-shift” into bulk water. Second, the protocell stage started with a substage of “pseudo-protocells”, with functional molecules catalyzing template-replication and metabolism, but still missing the function involved in the synthesis of membrane components, the emergence of which would lead to a subsequent “true-protocell” substage. Third, the initial unstable membrane, composed of prebiotically available fatty acids, should have been superseded quite early by a more stable membrane (e.g., composed of phospholipids, like modern cells). Additionally, the membrane-takeover probably occurred at the transition of the two substages of the protocells. The scenario described in the present study should correspond to an episode in early evolution, after the emergence of single “genes”, but before the appearance of a “chromosome” with linked genes.

Citation: Ma W, Hu J (2012) Computer Simulation on the Cooperation of Functional Molecules during the Early Stages of Evolution. PLoS ONE 7(4): e35454. https://doi.org/10.1371/journal.pone.0035454 Editor: Attila Szolnoki, Hungarian Academy of Sciences, Hungary Received: December 17, 2011; Accepted: March 16, 2012; Published: April 13, 2012 Copyright: © 2012 Ma, Hu. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The study was supported by the National Natural Science Foundation of China (No. 31170958, 30870660, 20927003, 90913013) and the National 973 Basic Research Program of China (No. 2010CB530500, 2010CB530503). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction According to the logic that “the simpler, the more possible to emerge from a non-life background”, life in the beginning should have been in some simple form (yet capable of Darwinian evolution). Hence, when it was revealed that RNA, acting as genetic material sometimes instead of DNA, could also act as functional (catalytic) molecules instead of proteins [1], [2], it began to be popularly believed that some early life forms were based solely on RNA, referred to as the “RNA world” [3]–[5]. An extreme version of this hypothesis states that the RNA-based life was just the earliest form (“RNA first”), re-emphasized by recent evidence concerning the plausibility of prebiotic nucleotide synthesis [6], [7]. Alternatively, the earliest life form may be based on some type of RNA-like polymer, in a pre-RNA world (“RNA later”) [4], [5]. For convenience, the present model was constructed and described in an “RNA first” view; however, similar conclusions may also be applicable for the molecular cooperation present in a pre-RNA world. For the “RNA first” view, it has long been proposed that the first functional RNA to emerging was a ribozyme catalyzing the template-directed copying of RNA [4], [5], which may spread in a nucleotide pool by favoring its own replication (called “RNA replicase”, here “Rep” for short). We have shown this plausibility by computer simulation assuming that the Rep could adopt a simple ligase form [8]. Alternatively, some ribozyme(s) catalyzing the synthesis of nucleotides (“nucleotide synthetase ribozyme”, here “Nsr” for short), by supplying building blocks around itself and thus favoring its own replication, may also have emerged first; the plausibility of this has also been shown by our simulation work [9]. No matter which ribozyme was first, it is interesting to see whether the two different functional RNAs, self-replicating independently, could coexist in the same system while competing for a limited source of raw materials and, moreover, cooperate in this “naked” stage (Fig. 1a). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. A scheme describing the cooperation of ribozymes in the early stages of the RNA world. Dots denote raw materials. “L-shapes” denote nucleotides. “Ball-sticks” denote amphiphiles. (a) RNA replicase (“Rep”, white) may cooperate with nucleotide synthetase ribozyme (“Nsr”, grey) in the “naked” stage. (b) Rep and Nsr may cooperate in the pseudo-protocell substage. (c) Rep, Nsr and amphiphile synthetase ribozyme (“Asr”, black) may cooperate in the true-protocell substage. The arrow shows that the amphiphiles may join the membrane of the protocell. https://doi.org/10.1371/journal.pone.0035454.g001 Evolution would eventually move into a protocell stage that included a membrane. The “cellular” coexistence/cooperation of Rep and Nsr is an interesting topic (Fig. 1b), especially in comparison with their cooperation in the naked stage. In the protocell stage, some ribozyme(s) synthesizing membrane components (“amphiphile synthetase ribozyme”, “Asr” for short) [10], [11] may have emerged, by contributing to the increase of the cellular space, thereby favoring the influx of additional raw materials and thus benefiting its own replication. The plausibility of this concept has been shown by our computer simulation work [12]. Protocells would then govern their own membrane synthesis, thereby becoming “true-protocells”. Consequently, it is quite interesting to incorporate the third ribozyme, i.e., Asr, into the system to determine whether it can coexist/cooperate with Rep and Nsr (Fig. 1c), and also to observe the behavior differences between “pseudo-protocells” (containing Rep and Nsr, but not Asr) and “true-protocells” (containing all three ribozymes). The importance of the coexistence/cooperation of self-replicating molecules in early-life evolution was suggested quite early, even before the proposal of the RNA world hypothesis. In the early 1970s, Eigen [13] suggested that non-enzymatic template replication would have low fidelity and could only sustain information carried by short nucleic acids (<50 nt) transferring from generation to generation. Short self-replicating nucleic acids without (enzyme-like) functions would compete with each other, leading to the result that only the “fittest” species (in the sense of acting as a template) would survive. According to Eigen, the emergence of a larger genome would have to involve function-carrying molecules, i.e., proteinaceous enzymes. Therefore, he proposed that there should be some self-organizing system in early life, in which one of the short self-replicating nucleic acids, by its coded polypeptides, favored self-replication of the next, therefore finally forming a closed cycle, called a “hypercycle”. A key problem with this concept is how such a complicated system could emerge in the origin of life, especially when both transcription and translation machineries need to be considered. Following the identification of ribozymes [1], [2] and the proposal of the RNA world hypothesis [3], it appeared that short self-replicating RNAs may act as function-carriers themselves. Consequently, the collective system proposed by Eigen could be replaced by a corresponding system purely based on RNA, which has a significantly reduced complexity and would be more likely to emerge in the origin of life. This is not the sole implication of the RNA world on Eigen's theory. If, as shown by computer simulation studies, short self-replicating RNAs could act as replicases (if adopting a simple ligase form, they may be shorter than 50 nt) [8] and could evolve towards higher efficiency and fidelity [14], the need to overcome Eigen's error threshold by the coexistence/cooperation of functional self-replicating species should no longer exist. However, the emergence of the molecular coexistence/cooperation remains important, because it appears to be the most natural path to complexity and efficiency after the emergence of individual functional RNAs. After Eigen's work, some alternative mechanisms of coexistence/cooperation of functional self-replicating molecules (referred to as “replicators” following Dawkins [15]; just “RNA” in the view of the RNA world) were proposed. For example, the metabolic coupling model suggested that replicators catalyzing intermediate steps of monomer synthesis (i.e., Nsr-series) could coexist/cooperate [16]–[20], and the stochastic corrector model (SCM) emphasized the role of group selection of functional replicators within a protocell that was dependent on the protocell division [21]–[23]. The models approached reality and in some aspects are better than the hypercycle model (for a review, see [24]). However, these studies are still limited. In particular, only a little attention has been paid to the coexistence/cooperation of molecules with different (unrelated) functions (e.g., it was suggested that Rep may appear from parasites of the Nsr-series system [18]). Noticeably, the synthesis of membrane components, as an important function of the protocell, i.e., Asr, has never been considered. Additionally, there was negligible parallel exploration of the naked and protocell systems (a recent study [25] is an exception, but this study only focused on the interaction of Rep and parasites, without explicitly taking into consideration other functions). In our previous simulation work, we have built explicit explanations describing the origin of individual functional RNA species, i.e., Rep [8], Nsr [9] and Asr [12]. The next stage in our study is the exploration (using similar models) of the plausibility of the coexistence/cooperation of these ribozymes with different (unrelated) functions, which represents a natural way towards complexity/efficiency in early evolution. In particular, as Rep functions in template-directed replication, Nsr functions in the metabolism of genetic and functional materials, and Asr is involved in the metabolism of the membrane, it can be concluded that the three functional RNA species included in the present study cover the fundamental requirements for the so-called minimal protocell [26]–[32]. Overall, the aim of this study was to show whether self-replicating RNA species, with functions at these different aspects, could coexist/cooperate (in both naked and protocell forms) while competing for common resources in the same system. The study also provides insights into the possible conditions and the history of this early evolution, after the advent of single genes yet before the emergence of chromosomes with linked genes.

Methods The simulation is based on a Monte-Carlo model, in which each event in the system may occur with some numerical probability in a time (Monte-Carlo) step. The mechanism of the model is the same as our previous work studying the behaviors of Rep [8], Nsr [9] and Asr [12]. An N×N grid was used for the system, with toroidal topology to avoid edge effects. Only molecules within the same “grid room” could interact. A “grid room” represents a square in the grid. It is referred to as a “grid cell” in traditional stochastic cellular automaton. Here, to avoid its confusion with protocells in the model and to emphasize it as a space for molecules to reside, we call it a “grid room”. In a time step, each event may occur with some probability, as explained below (also see Fig. 2). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 2. Events occurring in the model and their associated probabilities. Solid arrows represent chemical events and dashed arrows represent other events. (a) The events occurring in a grid room. Legends: Np, nucleotide precursor; Nt, nucleotide (A, U, C, or G); Ap, amphiphile precursor; Am, amphiphile. This version is for the true-protocell system. For the pseudo-protocell system, the events concerning Asr would not occur. For the naked system, the events concerning amphiphilic molecules and their precursors would not occur; there is no membrane at the edge of the grid room; nucleotides and RNA may also move to an adjacent grid room (see Table 1, note b). (b) Events concerning the behaviors of the protocells. When a protocell move to an adjacent (top, down, left, or right) naked grid room, the protocell would push away molecules in that room. When a protocell divides, amphiphiles on the membrane and molecules in the protocells would be distributed randomly between the two offspring protocells. One of the offspring protocells would occupy an adjacent naked grid room and push away molecules in that room. https://doi.org/10.1371/journal.pone.0035454.g002 A nucleotide precursor may transform to a nucleotide (randomly as A, U, C, or G) with the probability P NF (see Table 1 for a description of the abbreviation and those appearing in the following). A nucleotide may decay into a nucleotide precursor with P ND . A nucleotide residue at the end of an RNA chain may decay with P NDE . A nucleotide may be ligated with another nucleotide or an RNA chain at its end with P RL . A phosphodiester bond within an RNA chain may break with P BB (the probability is multiplied by F BO for RNA out of the protocells). An RNA may turn into a template (unfolding) with P RTT and attract nucleotides or oligomers with P AT by base-pairing (the probability of false base-pairing at each residue site is P FP ; for an oligomer, the base-pairing test is applied for all its residues). Nucleotides and oligomers aligned adjacently on the RNA template may be ligated to each other with P TL (template-directed ligation). The substrates or the products on the RNA template can dissociate if base pairs between them can separate (each base pair may separate with P SP ). PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Parameters used in the Monte Carlo simulation. https://doi.org/10.1371/journal.pone.0035454.t001 An amphiphile precursor may transform to an amphiphile with P AF . Amphiphiles (with a lower limit of quantity L AM ) may assemble into membrane at the edge of a grid room with P MF , encompassing molecules within it and forming a “protocell”. A free amphiphile may decay into an amphiphile precursor with P AD , whereas one within a protocell membrane (not within the protocell, just on the membrane) may decay with P ADM . A free amphiphile may join a protocell's membrane with P AJM , whereas an amphiphile within a protocell's membrane may leave it with P ALM . Nucleotides and RNA are assumed to be impermeable, whereas a nucleotide precursor and an amphiphile precursor may diffuse across the membrane with P NPP and P APP , respectively. A protocell may divide into two with P CD and two adjacent protocells may fuse into one with P CF . A protocell may break (into free amphiphiles) with P CB . An RNA containing a characteristic domain (presumed arbitrarily) may function as a corresponding ribozyme: Rep, Nsr, or Asr. However, if the length of the RNA equals or exceeds twice the characteristic domain, it is deemed that the “correct” structure would be interfered by the redundant residues and the RNA would not act as the ribozyme. A Rep molecule may bind onto an RNA template with P RB and drop from the template with P RD . If there is a Rep on the template, the template-directed ligation may occur with P TLR . However, if one or both base pairs flanking the ligation site are false, the ligation will not occur unless another probability, P FLR , is satisfied. In the model, P FP and P FLR are two parameters associated with the replication fidelity. The replication fidelity will increase when P FP and P FLR are small. An Nsr molecule may catalyze the formation of a nucleotide from a nucleotide precursor with P NFR , and an Asr molecule may catalyze the formation of an amphiphile from an amphiphile precursor with P AFR . Before the next time step, molecules and protocells in a grid room may move into adjacent rooms. A nucleotide, a nucleotide precursor, an amphiphile, or an amphiphile precursor may move with P MV , whereas a protocell may move with P MC . The factors F OP and F SI , as well as detailed assumptions concerning some events mentioned above have been explained in the notes of Table 1. Some considerations were taken into account to develop a logical setting of the numerical probabilities. Ribozymatic reactions should be much more efficient than corresponding non-enzymatic reactions, so P TLR >>P TL , P NFR >>P NF , and P AFR >>P AF . “Template-directed ligation” should be significantly more efficient than “random ligation”, so P TL >>P RL . Nucleotide residues in an RNA chain should be protected. Here, nucleotides within the chain are assumed to be unable to decay, whereas those at the end of the chain decay at a rate obviously lower than that of free nucleotides, i.e., P NDE <<P ND . Amphiphiles within membrane should be protected, so P ADM <<P AD . Owing to the self-assembly feature of amphiphilic molecules, P MF >>P CB and P AJM >>P ALM . The movement of molecules should be easier than protocells, so P MV >P MC . Other considerations may include: P BB may be at the same order as P RL , P ND >P NF , P AD >P AF , and P NPP ≤P APP . In a simulation case for the naked stage, nucleotide precursors in the quantity of T NPB were introduced in the initial step, and Rep and Nsr were inoculated at an early step. In a simulation case for the protocell stage, nucleotide precursors in the quantity of T NPB and amphiphile precursors in the quantity of T APB were introduced in the initial step, empty protocells were inoculated soon after the initial step, and then protocells containing ribozymes (Rep and Nsr for the pseudo-protocell substage; Rep, Nsr and Asr for the true-protocell substage) were inoculated at an early step. “Internal” events in the model, as described above, govern the whole dynamic process, step by step, occurring in the system.

Author Contributions Conceived and designed the experiments: WTM JMH. Performed the experiments: WTM. Analyzed the data: WTM. Wrote the paper: WTM JMH.