The larger the number of attractors, the smaller the chance of convergence. Compartmentalizing the reaction networks enables filtering out harmful modifications and therefore it is a prerequisite for accumulating potential beneficial 'adaptations', as demonstrated in [ 19 ]. We modelled compartments exactly as in Farmer et al.
The number of attractors is itself of interest, for they allow a protocell to have multiple pathways of autocatalysis and also to show molecular variability to respond to an environment. We approximated the number of attractors by fixing the reaction network and shuffling the chemical concentrations by choosing random pairs of species and swapping their concentrations several times in order to sample various initial conditions.
After shuffling, the network dynamics are run for some fixed period of time until an attractor is reached. Even if multiple attractors exist, stochastic division might not generate sufficient variation to allow transition between them. To test this, we also simulated the more realistic situation where the compartment enclosing the generative chemistry was allowed to grow for a fixed period of time, after which it was assumed to split into daughter compartments, whose molecules were sampled from a polyhypergeometric distribution of molecular contents in the parental compartment.
Now we arrive at the critical issue of evolvability, which can be first rephrased as the potential of a population of compartmentalized molecular networks with different attractors to respond to selection; that is, to transit between different attractors according to the fitness value assigned to each of them. As a preliminary test of evolvability, reaction networks were subjected to artificial selection. A small population of 10 compartments was isolated for a fixed generation period.
After this time the fitness of each compartment, defined as the total mass of non-food species present at the end of the growth phase just prior to division, was assessed and production of the next generation occurred by taking molecule propagules from compartments on the basis of fitness proportionate selection roulette-wheel selection with elitism [ 34 ]. This elitist selection was used to always sample at least one propagule from the individual with the highest rank in any given generation.
The results of the artificial selection experiment were confirmed in numerical simulations of natural selection. Thus, in each time step a randomly chosen compartment from the whole population is selected for growing at a rate which is a function of its chemical composition.
One offspring replaces the parent compartment and the other a randomly chosen one from the population. Selection for a specific target was implemented by multiplying the rates of all reactions by a selective advantage S if it matched the characteristic composition of the desired attractor. We tested the evolvability in all three previously described models - the original Farmer-type autocatalytic sets, networks with inhibition, and networks with random novel species produced by uncatalyzed reactions - according to the principles described above.
In the case of the original networks [ 12 ] the results were straightforward: they always have only one attractor Additional file 1 and selection is not possible. This was not surprising considering that these networks contain only one autocatalytic core Additional file 1.
Therefore, the conclusion immediately follows from our previous considerations: Kauffman's [ 11 ] original polymer chemistry when enclosed in a finite space will eventually crystallize into the same attracting network which can never ever be a Darwinian unit. Interestingly, this behaviour is analogous to conceptually similar models' [ 4 ] where the whole catalytic network forms inevitably only one viable core and so ultimately converges to only one attractor.
Therefore, one important conclusion to be derived from our work is that we can definitively discard all autocatalytic networks discussed so far in the literature as units of evolution in the Darwinian sense, with the possible exception of [ 19 ].
However, it has been suggested [ 1 ] that the inclusion of inhibition in the Farmer-type network should permit the formation of autocatalytic sets having complex dynamical attractors. To determine if this is so we also run simulations introducing strong non-competitive inhibition as indicated above. Interestingly, our results substantiate this speculation because molecular networks now exhibited multiple attractors, but when the growth-splitting process was implemented spontaneous transitions between them were rare.
When transitions did occur, they happened either periodically or chaotically Additional file 1. Rather surprisingly, the artificial selection experiment excluded networks with inhibition from candidates of units of evolution, since the population typically settled down into one equilibrium or fluctuated stochastically or periodically between attractors and so attractors typically could not be stably selected Additional file 1.
Instead, the internal dynamics of the growth-splitting process completely overrode any effect of selection. This provides a clear counter-example to the widely accepted claim that the existence of multiple attractors is sufficient to allow selectability; it is not.
The crucial modification to the model was to allow rare novel species to appear from the shadow. In the few cases of networks in which spontaneous addition of new species resulted in the ignition of a novel viable loop, and thus novel cores, there always existed multiple attractors see Figure 2.
Note that we did not simulate inhibitory reactions in this version of the model; while they are certainly relevant in applications closer to real chemistry, their inclusion would have made our results on viable loops more difficult to interpret. Analogous to the idea that 'attractors' in biological systems have different stabilities i. We intuitively suspected that selection would work in networks with novel viable loops, and this was indeed the case. Our results can be summarized as follows: while networks with the viable core have an implicit selective advantage due to their higher growth rate, and so constitute the majority of the population, a one percent selective advantage attributed to the absence of the core is enough to significantly reduce the proportion of networks with viable cores in the population Figure 5.
The reason for the selectability in this model is that a novel viable core results in a new and distinct attractor for the reaction network, and due to its autocatalytic properties enables a higher non-food mass growth rate. Hence, we already have the basic requirements for natural selection to happen: two entities that are growing exponentially at different rates and have different division times [ 36 ]. Since it is always possible to lose the viable core upon protocell fission a loss mutation that is simply a function of propagule size , there is a kind of 'mutation-selection' balance if no novel chemical species can invade from the shadow.
When rare reactions are allowed, novelty can arise by generation of new viable cores, and they can be removed by selection if they reduce the growth rate of the compartment. Between-compartment selection as shown in Figure 5 arises due to the effect a core has on the compartment level fitness. For example, the large core Figure 4 sustains more non-food mass in its core and its periphery, and this increases the growth rate of the compartment. In reality, each molecule of the core and its periphery may confer a host of compartment level effects, e.
Selectability of potentially coexisting attractors in a molecular network. Each dot corresponds to a compartment just prior to division. Top Due to its autocatalytic properties a viable loop enables a higher growth rate and therefore the network with the large viable loop characterized by 26 reactions and dividing after approximately 20 time steps constitutes the most frequent network type. In this case the original network without any viable loops is the most frequent.
It is important to note that there are two levels of autocatalysis in this system. Even if the internal organization of the network encapsulated by a protocell fails to be autocatalytic, the rule that after reaching the critical mass the compartment divides into two effectively ensures that such compartments will have a 'generation time' and the potential to grow exponentially.
Also autocatalytic cores grow exponentially. Hence there is autocatalysis at two levels: the level of molecules and that of compartments. The reproducing compartment without an enclosed autocatalytic network is not, however, a replicator, as it always assumes the same state and cannot sustain hereditary variation [ 28 ].
It should come as no surprise, our finding that independent viable autocatalytic cores embedded in a large molecular network can be considered as units of evolution, since the basic ingredients of differential growth rates and division times among potentially competing entities are fulfilled. The reader might, however, query that we have not properly addressed the issue of evolvability because no mention of heredity has been made.
To fully understand that this criticism does not apply here it is important to appreciate the implications of the core-periphery dichotomy in autocatalytic sets. This dichotomy can be translated into a kind of genotype-phenotype mapping in fully fledged biological systems and, interestingly, allows us to appropriately use the terms replication and reproduction despite the fact that we are dealing with an assembly of molecules [ 37 ]. Thus, the viable cores could be considered the units that replicate and, once transmitted to the offspring compartments after the parental compartment splits reproduces , they give raise to the same periphery; that is; there is a clear matching between a viable core 'genotype' and the periphery 'phenotype'.
Thus, our autocatalytic networks are capable of stably transmitting information across generations [ 17 ]. However, a viable core constitutes one bit of heritable information and therefore the number of possible selectable attractors is relatively small, meaning that autocatalytic networks may not be able to sustain open-ended evolution.
While we think this to be the case, the potential role of these autocatalytic networks as a route to nucleotide-based template self-replicating systems should not be underestimated. The chemical reaction networks show an intrinsic tendency to increase in complexity. Whenever novel spontaneous reactions occur, the number of possible uncatalyzed reactions also increases, opening up new possibilities for discovering viable cores genotypes and their corresponding peripheries phenotypes.
This 'cooptive evolution' [ 13 ] involves stepwise expansions into and retractions from the adjacent possible of reaction space [ 38 ]. It is important to note that we do not claim that the present work renders the RNA world obsolete. In fact several of the authors of the present paper have worked under the assumption that indeed there had once been an RNA world. But this does not mean two things: first, that the RNA world was "clean" probably it was not; other molecules, large and small, are likely to have been around and to have served even key functions , second, that reflexively autocatalytic networks could not have preceded the RNA world they may have been indispensable "scaffolds", sensu Cairns-Smith, [ 39 ] for its appearance.
Genetic takeover does not in principle require reverse translation or any similar esoteric process; one just needs room for stepwise innovation and improvement. Our results should be contrasted with the lipid world scenario [ 4 , 16 , 40 ] that so far has failed to offer models that would demonstrate a capacity for evolvability.
The problem is that the simplicity of the underlying chemistry in GARD lipid molecules are either in the assembly or not allows only as many reactions as there are different molecular species available in the environment. Moreover, the number of distinct lipid types cannot be too high, partly because of practical considerations, but also because increasing diversity implies increasing noise in compotype replication.
The restricted diversity of molecules and reactions means that the system will always quickly converge to the state set in stone by the underlying dynamical equations [ 4 ]. The only possibility left open for change is the addition or removal of lipid species. Pointing out that altering the food set of a reaction network modifies its dynamics, however, has no relevance for evolution.
A combinatorial chemistry like the polymer chemistry described in this article, on the other hand, provides an unlimited diversity of theoretically possible reactions originating from the same food set and a reasonable probability that a reaction network can discover novel cores in its shadow.
Also, the permanent incorporation of a new core will extend the shadow, opening up new possibilities. Therefore we argue that such a combinatorial chemistry or one with similar complexity is essential for even limited evolution. The complexity of a lipid world is overshadowed by the possibilities enabled by the outlined polymer chemistry, which itself is only a shadow of the world of template-replicating nucleic acids.
We stress that there is a crucial difference between small-molecule autocatalytic cycles such as the reductive citric acid cycle and reflexively autocatalytic sets of polymers. First, a family of polymers such as proteins can be synthesized by a small set of canonical chemical reactions, whereas the reductive citric acid cycle consists of chemical steps of various kinds cf.
Orgel [ 41 ] , thus the former can more readily be catalyzed by environmental i. Second, and more important, polymers can, due to their modular construction, show targeted and specific activity in catalytic task space.
The increased efficiency of catalysis carries over to resistance against side reactions [ 41 ] that constantly divert material from useful pathways. However, these facts merely change the probability of formation of viable cores in particular chemical systems, not the fact that Darwinian evolution is possible once they appear.
The remaining open issues are experimental and theoretical in nature. We need better models and, above all, relevant experiments. The systematic consideration of the experimental realization and evolvability of autocatalytic networks of small organics such as those of intermediate metabolism [ 2 , 19 , 29 , 31 ] require further scrutiny in the light of the proposed selectability principles. It is not farfetched to claim that an empirical scientific program implementing the sort of simple chemistry used in these models is worth pursuing.
The recent calculations of Amend and McCollom [ 42 ] indicate that amino acid production in ancient hydrothermal vents could have been thermodynamically favoured, providing a continuous supply of monomers for the hypothesized peptide network. Autocatalytic networks of peptides already have been synthesized by Ghadiri and Ashkenazy [ 21 ], although there a direct templating effect plays a crucial role.
Protein networks that do not employ templating are more difficult to realize, but several recent advances hint they might be possible. There exists a dipeptide that does catalyze ligation of peptides [ 43 ]. The folding and catalytic properties of "never before born" peptides is therefore an open experimental question that could be addressed with random peptide libraries - it is a project much to be sought, and we should make it clear that these experiments are now needed, and hopeful given the promising results cited above.
Not every aspect of a key proposal for the spontaneous emergence of dynamical chemical organizations can be scrutinized in a single paper. Here we restricted ourselves to three issues: i the probability of the nucleation of reflexively autocatalytic networks, as questioned e.
We think we have advanced promisingly with all three problems in the present work. Our work shows that autocatalytic sets as first devised by Dyson [ 9 , 10 ] and Kauffman [ 1 , 11 ] are theoretically possible despite previous criticisms and, perhaps more interesting, that chemical evolution in these systems can lead to the appearance of viable autocatalytic cores, thus opening the possibility for evolution by natural selection.
We have used an abstract chemistry not to avoid real chemistry, but to seek general principles. For example, selection between autocatalytic cores may even be a possibility in combinatorial inorganic systems evolving in iCHELL compartments [ 46 ].
Naturally, one cannot be satisfied with abstract toy chemistry for long. But there is always a first step, and the scenario outlined here should give us hope that it is worthwhile to explore the idea further. After all, the pre-template Darwinian dynamics of rare core production and selection described here - fundamentally different from the mechanism advocated by Kauffman [ 1 ] and dismissed by Eigen [ 18 ] - is the only viable proposal so far for how autocatalytic reaction networks could accumulate adaptations.
Following Farmer et al. Reversible, ligation condensation and cleavage reactions, catalyzed by another polymer, were modelled. Reactions were of the form:. No attempt was made to relate the structure i. Each molecule has a certain probability P of catalyzing each theoretically possible ligation-cleavage reaction, and the reactions in which a polymer will participate, along with the catalysts, are determined randomly.
The chemical kinetics approximates the behaviour of catalyzed reactions enclosed in a compartment. We assumed identical binding velocity for all intermediates.
Only the catalytic rates vary in magnitude from 10 to Food was present at initial concentration F c and added continuously at rate F input , and all molecular species have a first order decay rate k d. We typically used 10 -5 M as a minimum concentration threshold below which no molecule of the species exists in the reactor for more details see Additional file 1.
In order to address Lifson's criticism [ 26 ], the parameter P was replaced by two independent probabilities: the probability P' that the molecule is a catalyst, and the probability P" that a molecule catalyzes a given reaction. In models including inhibition, when a new molecular species is first produced in a manner analogous to the determination of catalytic reactions it is determined which other species in the reactor the new species will inhibit with probability K , and which existing species will poison the new species also with probability K.
The probability of inhibition varied from 0 to 1. In the novel species model uncatalyzed reactions among available molecular species took place with a low probability the concentration of any molecular species had a probability ranging from 0 to 0. The three versions of the chemical model original, inhibition and with novel species , all confined into compartments, were subjected to the same tests in order to assess their evolvability.
We approximated the number of attractors in chemical networks by fixing the reaction network and shuffling the chemical concentrations by choosing random pairs of species and swapping their concentrations several times in order to sample various initial conditions. After shuffling, the network dynamics are run for time steps that appeared enough to reach a new attractor. Even if multiple attractors exist, stochastic division might not generate sufficient variation to transition between them.
To test this, compartments enclosing the generative chemistry were allowed to grow for a fixed period of time after which compartment splitting was modelled by taking a polyhypergeometric distribution of molecular contents. Typically the propagule size was molecules to allow sufficient variability, also permitting loss of species upon division. In addition, a low probability of spontaneous appearance in low copy number of species that already exist in the platonic reaction network but have gone below threshold concentration was assumed, allowing the re-emergence of lost species.
In the artificial selection experiment a population of 10 compartments was isolated for a fixed generation period. After this time the fitness of each compartment was assessed and production of the next generation of 10 new compartments occurred by taking molecule propagules from compartments on the basis of fitness proportionate selection; i. Elitism was used to always sample at least one propagule from the individual with the highest rank in any given generation.
Two different targets of selection were imposed. First, fitness of a compartment was defined as the total mass of non-food species sum of the concentration multiplied by length for all non-food species present in a compartment at the end of the growth phase, just prior to division. Second, the fitness was defined as the reciprocal of this value. As the last step, the response of the chemical networks to natural selection was tested in numerical simulations.
Selection for a specific target was implemented by multiplying the rates of all reactions by a selective advantage S set to 1. Google Scholar. Shapiro R: A simpler origin for life. Proceedings of the National Academy of Sciences. Bulletin of the Museum of Comparative Zoology.
Szathmary E: Simple growth laws and selection consequences. Journal of Molecular Evolution. Dyson FJ: Origins of Life. Journal of theoretical biology. Physica D. Article Google Scholar. Fernando C, Vasas V: Cooptive evolution of prebiotic chemical networks. Edited by: Seckbach J, Gordon R. However, some cosmologists believe that the proto-solar system was initially a partially ionized gas, in which case the notion of equilibrium condensation would not be valid.
Sometime within million years of Earth's birth, life arose on its surface and biological evolution began. Eventually, the death of the Sun may be accompanied by an ejection of matter back into the interstellar medium that spawned it. According to this scenario, the origin and evolution of life on Earth were and will continue to be inextricably bound to the evolution of both the Sun and Earth. It is somewhat ironic that life arose on Earth, a planet that, relative to the Sun, is severely depleted in the volatile elements that make up organic chemistry: hydrogen, carbon, nitrogen see table 1.
On the other hand, the chemistry of the cosmos seems to be dominated by these elements. From this knowledge springs the conviction that organic chemistry constitutes an integral and fundamental part of cosmochemistry, and from this comes the anticipation that, despite the seeming improbability TABLE 1.
Element Sun Earth Biosphere. Hydrogen 94 0. With this introduction we shall proceed to a discussion of interstellar clouds, comets, outer planets, asteroids as represented by meteorites, and the primitive Earth, and consider the organic chemistry of these various environments. A roster of molecules observed in the interstellar medium is given in table 2.
The more complex molecules triatomic or larger occur in the dense clouds. The bulk composition of the interstellar gas is presumed to reflect cosmic elemental abundances. The dust in interstellar clouds is not well characterized; there is evidence to suggest the presence of ice, silicates, graphite, macromolecular organic compounds, and mixtures of these ingredients.
The dust and molecules may have come from several sources: some of it may be a remnant of nebula condensation and solar-system formation, that is, material ejected into the interstellar medium by the T-tauri stage of stars, and some of it may have been ejected from the dense atmospheres of giant stars. Estimates of the lifetimes of dense clouds before gravitational collapse exceed estimates of the lifetimes of molecules in the gas phase before freezing out on dust grain surfaces.
Therefore, the fact that we observe interstellar molecules in the gas phase indicates a continuous production mechanism within the clouds themselves. In formulating a production mechanism, one must consider the environment in which it occurs. The temperatures are very low, 3 to K, which means that chemical reactions in the clouds except some reactions of hydrogen atoms must occur with essentially zero activation energy. In addition, the extremely low concentrations of molecules mean that all collisions between them and therefore chemical reactions are binary, that is, they involve only two species.
These and other constraints have led to a model for the synthesis of interstellar molecules in dense clouds in which reactions are initiated by collisions of ubiquitous, high-energy, cosmic-ray particles with H 2 and He. Reactive ionic species are generated Inorganic Organic. SO Sulfur monoxide. OCS Carbonyl sulfide. Some Several types of reactions are shown in table 3. In addition, chemical reactions may occur on dust grain surfaces that act as "collectors," and these would involve the recombination of free radicals, a type of reaction requiring little or no activation energy.
In addition, atoms and free radicals may react with the grain itself. Examination of the list of compounds in table 2 leads to two important observations.
First, the compounds are chemically diverse and structurally complex. Second, many of them are known to be important intermediates in the production of organic matter in abiotic synthesis experiments e. Clearly, the interstellar environment, as exotic and as seemingly inimical to chemical reactions as it may appear at first consideration, nonetheless exhibits a rich chemistry that manifests itself in the production of organic compounds that, for the most part, are familiar from terrestrial experience.
TABLE 3. These possibilities stem from the idea that all solar-system matter had a common origin in an interstellar cloud of dust and molecules. To the extent that comets and carbonaceous meteorites contributed mass to early Earth and arrived at the surface intact , interstellar organic compounds could have survived to take part in subsequent chemical evolution. Comets occupy an especially interesting place in models of solar-system origin and evolution.
They may have been a partial source of planetary atmospheres, and they are believed to have been the building blocks for the rocky cores of the outer planets.
Present understanding places the origin of comets in the outer regions of the primitive solar nebula in and beyond the space now traversed by the giant planets. The components of a comet observable within several astronomical units of the Sun include the nucleus, the coma, and the tail fig. According to the "dirty ice" model, comet nuclei consist of simple and complex organic molecules and meteorite-like dust and rock embedded in a matrix of frozen water and possibly solid CO 2.
In the coma, interactions of the parent compounds with solar radiation can lead to physical and chemical processes that result in the partial-to-complete breakdown of the so-called parent molecules.
Ion-molecule reactions analogous to those occurring in interstellar clouds probably play an important role. The neutral daughter products are observed in the coma, while the positively charged ones are observed in the tail. According to an alternative view, all the observed species already exist in the nucleus and are simply released directly into the coma by evaporation.
These two possibilities arc summarized in figure 3. A third possibility is that both parent molecules and simpler species are released into the coma where they undergo reactions to yield the Figure 2. Major features of a comet. The distance scale is logarithmic. Ions are observed in the tail, neutral species in the coma. In addition to the species indicated in figure 2, metallic elements Fe, Si, Mg, Ca, Ni, Na, Cr have been detected in spectroscopic studies of meteor showers associated with comets.
The relative abundances of these elements suggest similarities between the chemical compositions of cometary dust and carbonaceous meteorites. The comet nucleus is thought to be small, typically km in diameter, but no direct observations have yet been made. It appears as a small point of light embedded within the bright, large, and extensive coma. The mass of the nucleus could range from 10 15 to 10 18 gm. The light from the visible coma and tail is emitted by atoms and molecules that have interacted with solar radiation.
The size of the coma is remarkable, perhaps greater than 10 5 km in radius. The tail, composed of dust grains and ionic molecules, is even larger, possibly exceeding 10 7 km in some cases.
When comets become visible in the inner Solar System, they may be spatially the largest objects in the sky. As mentioned above, comets are believed to be material condensed and accreted at the outer edge of the primitive solar nebula. Thus a relationship may exist between comets and interstellar molecules; if one compares the molecules observed in comets fig.
Figure 3. Production of observed cometary molecules by direct evaporation from the nucleus or by evaporation of parent molecules followed by their interactions with solar radiation. Both populations contain cyanide derivatives with the CN group, and comet species can be produced by fragmentation of interstellar molecules. It has also been suggested that carbonaceous meteorites, which are rich in various forms of the volatile elements and organic matter, are remnants of volatile-depleted and moribund comets.
If comets do not contain relatively unaltered interstellar matter, and if they formed at the outer edge of the solar nebula where temperatures were low enough to condense gases such as carbon dioxide and water, then the presence of parent organic molecules in comets is difficult to understand. There is no widely accepted model for chemical reactions in the solar nebula that could yield the chemistry of comets.
Indeed, without direct observations of the nucleus, our knowledge of comet chemistry is exceedingly sparse and model-dependent. Since comets are poorly understood and may represent a chemical evolutionary link between the primitive solar nebula and the interstellar medium, their direct study by space probes constitutes a high-priority objective for many space scientists.
From the outer regions of the Solar System where comets originated, we move sunward to consider organic chemistry on Jupiter. Spectroscopic observations made by astronomers, theoretical considerations, and, more recently, direct study by space missions provide the basis for current models of Jupiter. The planet has approximately a solar-composition atmosphere consisting primarily of hydrogen and helium with minor to trace amounts of methane, ammonia, water, hydrogen sulfide, ethane, acetylene, phosphine, carbon monoxide, arsine, and the noble gases.
The planet's compositional similarity to the primordial nebula makes it a critical object for cosmological study. Planetary processes that prevailed soon after its origin are probably still occurring now.
The model of the environment of the upper and observable Jovian atmosphere is depicted schematically in figure 4. Sunlight and extraplanetary particles arc represented as entering at the top of the atmosphere. The locations of a haze layer and the various observed and postulated cloud layers are also shown, as are the directions of motion of the latter thick arrows according to recent meteorological models. The depths in terms of temperature and pressure to which sunlight of various wavelengths penetrates into the atmosphere are indicated by the vertical arrows.
The presence of gas constituents is also shown by arrows, starting at about the maximum altitude in the atmosphere where they may occur. The stratification of some atmospheric components results from the formation of their solid or liquid condensates at different temperatures and altitudes. The jagged lines through the clouds indicate lightning flashes.
The distinct coloration of Jupiter's cloud cover and the variability of its patterns with time have been observed for over a century. These were the first indications of the occurrence of disequilibrium processes in the atmosphere. In , Urey first suggested that complex organic molecules might be responsible for the cloud colors.
Since then the hypothesis that Jupiter is at an advanced stage of organic chemical evolution has been widely promulgated.
Support for this view has been marshaled from observations that complex organic compounds and colored organic polymers are produced when gaseous components life those in the Jovian atmosphere are subjected in laboratory experiments to electric discharges, thunder shock waves, high-energy proton irradiation, or ultraviolet irradiation.
On the basis of theoretical models of Jovian atmospheric photochemistry, however, the contrasting view that the colors are attributable to inorganic substances photochemically Figure 4.
Summary of features in a model of Jupiter's atmosphere. No data yet obtained from planetary observations can verify either viewpoint to the exclusion of the other. The hydrocarbons observed on Jupiter-acetylene C 2 H 2 and ethane C 2 H 6 -are believed to be produced by the interaction of sunlight with methane high in the atmosphere.
There is also good reason to believe that acetylene is synthesized mainly during thunderstorms. The origin of the carbon monoxide is not clear. Two processes have been suggested which may act separately or jointly: an upwelling of material from deep in the [ 33 ] atmosphere, where at high temperatures carbon monoxide is thermodynamically stable, or reactions of atmospheric methane with oxygen atoms injected into the upper atmosphere from extraplanetary sources.
Images from the Voyager mission have shown lightning flashes on Jupiter near the ammonia clouds. Although the occurrence of lightning does not prove that the colored material is organic matter, it supports the view that organic matter is produced on Jupiter as it is in laboratory experiments designed to simulate Jovian phenomena.
This would not have been possible without DNA-programmed chemistry. There are many other types of single-shot selections that are highly valuable, for example selection for ligands to receptors that are overexpressed in intact cells. Finally, the evolutionary cycle made it possible to generate exceptionally fit hybrids by recombination of enriched genes between generations. This enabled the "genetic algorithm" for functional optimization, a process that has proved extremely powerful in the context of directed protein evolution see e.
Although library diversification between generations was not necessary in our experiment, because the limited library diversity was oversampled, it will be essential for complex chemical spaces that cannot be adequately sampled in a single generation.
Natural products are the fruits of bio-combinatorial chemistry and chemical evolution that occurred over billions of years. Enzymes carried out the small-molecule synthesis and a diverse array of metabolites served as building blocks. Here we have demonstrated a gene translation system that accommodates extremely large genetic codes, making it possible to reenact the chemical-evolution process in a test tube.
With unlimited synthetic possibilities and universal building-block alphabets, evolution is sure to yield surprises. Hopefully, these will include molecules with remarkable functional properties, akin to the natural products which inspired this work. Louis, MO. DNA-programmed combinatorial chemistry derives from the split-pool technique for synthesis of combinatorial small-molecule libraries on polystyrene beads.
There are a few key differences. First, a short DNA fragment the "gene" replaces each polystyrene bead, and the DNA fragment acts as the solid support. A molecule is synthesized at the end of a linker attached to the DNA fragment. For the experiments reported here, there is only a single copy of the small molecule associated with each fragment. Chemical reaction steps are performed while the DNA fragment is absorbed onto anion-exchange beads, so that conventional filter-based strategies for solid-phase synthesis can be used.
Second, the splitting step is fundamentally different. Whereas the polystyrene bead supports are split randomly into different reaction vessels in a conventional split-pool approach, the DNA-fragment supports are split deterministically into different reaction vessels in the DNA-programmed approach. Specifically, each DNA fragment is routed to a single position of a microcolumn array the "anticodon array" because a short sequence within the DNA fragment hybridizes to a complementary oligonucleotide immobilized on one microcolumn in the array.
The routed DNA fragments are then transferred from the microcolumns into the corresponding wells of a well filterplate.
The filterplate contains anion-exchange resin beads that bind to and retain the DNA fragments. The wells of the filterplate then serve as independent chemical reaction vessels for coupling reactions. A different synthon is used as the reactant in each well. Thus, because the nucleotide sequence of each DNA fragment determines the reaction vessel it winds up in, the nucleotide sequence also determines the chemical transformation to which the fragment will be subjected.
The process of routing and chemical coupling is repeated for each step in a multi-step synthetic pathway. For a four-step synthesis, for example, four distinct sub-sequences within each DNA fragment determine the four microplate positions that the fragment will adopt during the library synthesis.
By determining these microplate positions, each DNA fragment programs a sequence of four chemical transformations. This synthetic sequence produces the small molecule to which the fragment is ultimately attached. Although these solvent conditions are incompatible with DNA solubility, the reactions proceed efficiently while the DNA is absorbed onto anion exchange beads.
The workflow for programmed library synthesis, comprising a series of DNA-directed library splitting steps followed by chemical coupling steps, was carried out as previously described [ 19 , 22 ]. The anticodon array with bound DNA was then mounted into a well adapter device [ 23 ] which uses rubber gaskets to form an isolated liquid channel above and below each array feature.
Following the synthetic steps, the filter plate was placed on top of a well polypropylene microtiter plate, and DNA was eluted from the DEAE support by application of a high salt buffer 1. The eluted DNA was pooled, concentrated and buffer exchanged into hybridization buffer using a centrifugal filter device with a 10, Da molecular weight cut-off GE Healthcare. The sample was diluted to 3 ml with hybridization buffer and applied to another anticodon array.
The two halves of the library were concentrated with n-butanol extractions, precipitated with isopropanol, and subjected to a kinase-substrate selection or a control selection, respectively. After incubation, the crude reactions were diluted 1.
The library was resuspended in The beads were eluted, and the purification process was repeated a second time on a fresh MACS column. Paired-end bp reads were obtained on a MiSeq Illumina Sequencer.
First, a string search on the fastq MiSeq files was used to locate constant-region sequences Z a -Z f. The 20 base-pair blocks adjacent to each constant region were excised and saved. The forward reads gave sequences for the first four codons A-D , and the reverse reads gave reverse-complement sequences for the last five codons B-E.
Redundant forward and reverse reads were obtained for the three central codons B-D. The 20mer blocks were converted into codon numbers using a direct string comparison to each of the possible codon sequences. In order for a codon number assignment to be made, at least 18 bases had to match between the observed 20mer and the reference codon sequence. Paired reads that any gave contradictory assignment at codons B-D were discarded. The raw list of codon sequences was sorted, and the reads were summed, to generate a non-redundant list of codon sequences and the number of times that each sequence was observed in the data.
The codon-sequence data were then split into a kinase-selected block and a mock-selected block based on the identity of the E codon. The codon frequencies at each coding position in the mock-selected library were calculated, and the fractional abundance of each four-number codon sequence in the mock library was approximated as the product of the frequencies of its constituent codons. The fold-enrichment ratios of genes with two or more reads in the kinase-selected library were calculated as the ratio of the gene's fractional abundance in the kinase-treated population relative to its apparent fractional abundance in the mock-treated control population.
For the analysis with a reduced number of reads, a subset of the total reads was selected randomly. The calculations were repeated 50 times with different random subsets, and the 50 receiver-operator characteristic curves were averaged. For the analysis based on a two codon per amino acid genetic code, synonymous codons were paired randomly. The calculations were repeated 50 times with different random pairings, and the 50 receiver-operator characteristic curves were averaged.
The distribution of log fold-enrichment ratios expected by chance from a finite DNA sequencing sample was computed based on 3 million gene reads, an assumed enrichment of , for all RRSFL-encoding genes, and the prior codon abundances observed in the control gene population. Candidate peptides were synthesized as C-terminal amides. Phosphorylation of the peptides by protein kinase A was measured using a P81 phosphocellulose filter binding assay with 32 P-ATP [ 46 ].
Substrate concentrations were normalized to the kemptide concentration by coupling each peptide to fluorescamine, separating the peptide from unreacted fluorescamine on a high-pressure liquid chromatography column, and integrating the signal from a fluorescence detector.
Substrate concentrations were additionally verified by measuring the radioactive signal following exhaustive peptide phosphorylation overnight incubation with 40 nanomolar PKA.
Initial phosphorylation rates were measured at peptide concentrations between 0. In all cases, the observed initial rates increased linearly with peptide concentration. The data were used to fit a second order rate constant relative to the kemptide standard. Control peptide-DNA conjugates were spiked into an excess of background DNA, and then subjected to the biochemical selection that enriches for protein kinase A substrates. The fractional abundances before and after selection were measured by quantitative PCR.
Sequence logos for the two classes of peptides encoded by highly enriched genes selected for PKA substrates. Peptides were weighted by their enrichments and sequence logos were prepared using weblogo.
Bottom : Substrate class matching the consensus sequence for PKA. Codon abundance in the initial DNA population is plotted against codon abundance in the fourth generation mock-selected population. Codons specifying an amino-acid coupling step are green.
Codons specifying a blank no chemistry step are blue. True fitness rank is the rank of the encoded peptide. The peptide ranks were determined by summing reads over all of the genes that encoded each peptide. Twenty extreme outliers with spurious single-gene enrichments are evident.
The corresponding peptide products all include two or more blank building blocks. Riordan and Erik Wulff for reading the manuscript, and Rebecca M. Weisinger for helpful discussions while the project was being planned. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract The first demonstration that macromolecules could be evolved in a test tube was reported twenty-five years ago.
Introduction Evolution, the change in the inherited characteristics of biological populations over successive generations, accounts for the diversity of life on earth. Download: PPT. Results Design of the Chemical Evolution Experiment DNA-programming of an n-step chemical synthesis in microplate format can accommodate a library complexity of n. Table 1. Population Behavior Under Selective Pressure To estimate the discovery power of our directed evolution system, we analyzed the data as though each codon represented a distinct amino acid.
Discussion Ultimately, the purpose of directed evolution is to identify functional molecules hidden within large chemical spaces. DNA-programmed Combinatorial Chemistry DNA-programmed combinatorial chemistry derives from the split-pool technique for synthesis of combinatorial small-molecule libraries on polystyrene beads.
Supporting Information. S1 Fig. S2 Fig. Related to Table 1 : Consensus sequences of evolved substrates. S3 Fig. Related to Fig 4 : Concordance of codon abundance. S4 Fig. Related to Fig 4 : Concordance of single-gene enrichment rank with true fitness rank. S1 File. Supporting Experimental Procedures with six supplemental references. References 1. Smith GP Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface.
Science — Gene 83— Nature — Nat Methods 4: — Cold Spring Harb Perspect Biol 4. View Article Google Scholar 8. Annu Rev Chem Biomol Eng 2: 53—
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