Constraint Closure Drove Major Transitions in the Origins of Life.

Life is an epiphenomenon for which origins are of tremendous interest to explain. We provide a framework for doing so based on the thermodynamic concept of work cycles. These cycles can create their own closure events, and thereby provide a mechanism for engendering novelty. We note that three significant such events led to life as we know it on Earth: (1) the advent of collective autocatalytic sets (CASs) of small molecules; (2) the advent of CASs of reproducing informational polymers; and (3) the advent of CASs of polymerase replicases. Each step could occur only when the boundary conditions of the system fostered constraints that fundamentally changed the phase space. With the realization that these successive events are required for innovative forms of life, we may now be able to focus more clearly on the question of life's abundance in the universe.

Limited Sequence Diversity Within a Population Supports Prebiotic RNA Reproduction.

The origins of life require the emergence of informational polymers capable of reproduction. In the RNA world on the primordial Earth, reproducible RNA molecules would have arisen from a mixture of compositionally biased, poorly available, short RNA sequences in prebiotic environments. However, it remains unclear what level of sequence diversity within a small subset of population is required to initiate RNA reproduction by prebiotic mechanisms. Here, using a simulation for template-directed recombination and ligation, we explore the effect of sequence diversity in a given population for the onset of RNA reproduction. We show that RNA reproduction is improbable in low and high diversity of finite populations; however, it could robustly occur in an intermediate sequence diversity. The intermediate range broadens toward higher diversity as population size increases. We also found that emergent reproducible RNAs likely form autocatalytic networks and collectively reproduce by catalyzing the formation of each other, allowing the expansion of information capacity. These results highlight the potential of abiotic RNAs, neither abundant nor diverse, to kick-start autocatalytic reproduction through spontaneous network formation.

Mineral surfaces select for longer RNA molecules.

We report empirically and theoretically that multiple prebiotic minerals can selectively accumulate longer RNAs, with selectivity enhanced at higher temperatures. We further demonstrate that surfaces can be combined with a catalytic RNA to form longer RNA polymers, supporting the potential of minerals to develop genetic information on the early Earth.

Spontaneous advent of genetic diversity in RNA populations through multiple recombination mechanisms.

There are several plausible abiotic synthetic routes from prebiotic chemical materials to ribonucleotides and even short RNA oligomers. However, for refinement of the RNA World hypothesis to help explain the origins of life on the Earth, there needs to be a manner by which such oligomers can increase their length and expand their sequence diversity. Oligomers longer than at least 10-20 nucleotides would be needed for raw material for subsequent natural selection. Here, we explore spontaneous RNA-RNA recombination as a facile means by which such length and diversity enhancement could have been realized. Motivated by the discovery that RNA oligomers stored for long periods of time in the freezer expand their lengths, we systematically investigated RNA-RNA recombination processes. In addition to one known mechanism, we discovered at least three new mechanisms. In these, one RNA oligomer acts as a splint to catalyze the hybridization of two other oligomers and facilitates the attack of a 5'-OH, a 3'-OH, or a 2'-OH nucleophile of one oligomer onto a target atom of another. This leads to the displacement of one RNA fragment and the production of new recombinant oligomers. We show that this process can explain the spontaneous emergence of sequence complexity, both in vitro and in silico.

Coupled catabolism and anabolism in autocatalytic RNA sets.

The ability to process molecules available in the environment into useable building blocks characterizes catabolism in contemporary cells and was probably critical for the initiation of life. Here we show that a catabolic process in collectively autocatalytic sets of RNAs allows diversified substrates to be assimilated. We modify fragments of the Azoarcus group I intron and find that the system is able to restore the original native fragments by a multi-step reaction pathway. This allows in turn the formation of catalysts by an anabolic process, eventually leading to the accumulation of ribozymes. These results demonstrate that rudimentary self-reproducing RNA systems based on recombination possess an inherent capacity to assimilate an expanded repertoire of chemical resources and suggest that coupled catabolism and anabolism could have arisen at a very early stage in primordial living systems.

Spontaneous Covalent Self-Assembly of the Azoarcus Ribozyme from Five Fragments.

Spontaneous covalent assembly of short RNA fragments has been proposed as a plausible prebiotically relevant pathway to a self-reproducing system. We previously showed that the Azoarcus group I intron could self-assemble from four RNA fragments. Here, we extended this fragmentation to five RNAs that averaged <40 nucleotides in length. We optimized this reaction and showed that a dehydration-rehydration sequence was the most effective means to date to shift the self-assembly equilibrium from reactants to products.

Prebiotic RNA Network Formation: A Taxonomy of Molecular Cooperation.

Cooperation is essential for evolution of biological complexity. Recent work has shown game theoretic arguments, commonly used to model biological cooperation, can also illuminate the dynamics of chemical systems. Here we investigate the types of cooperation possible in a real RNA system based on the Azoarcus ribozyme, by constructing a taxonomy of possible cooperative groups. We construct a computational model of this system to investigate the features of the real system promoting cooperation. We find triplet interactions among genotypes are intrinsically biased towards cooperation due to the particular distribution of catalytic rate constants measured empirically in the real system. For other distributions cooperation is less favored. We discuss implications for understanding cooperation as a driver of complexification in the origin of life.

Life's Late Digital Revolution and Why It Matters for the Study of the Origins of Life.

The information contained in life exists in two forms, analog and digital. Analog information is manifest mainly in the differing concentrations of chemicals that get passed from generation to generation and can vary from cell to cell. Digital information is encoded in linear polymers such as DNA and RNA, whose side chains come in discrete chemical forms. Here, we argue that the analog form of information preceded the digital. Acceptance of this dichotomy, and this progression, can help direct future studies on how life originated and initially complexified on the primordial Earth, as well as expected trajectories for other, independent origins of complex life.

Topological and thermodynamic factors that influence the evolution of small networks of catalytic RNA species.

An RNA-directed recombination reaction can result in a network of interacting RNA species. It is now becoming increasingly apparent that such networks could have been an important feature of the RNA world during the nascent evolution of life on the Earth. However, the means by which such small RNA networks assimilate other available genotypes in the environment to grow and evolve into the more complex networks that are thought to have existed in the prebiotic milieu are not known. Here, we used the ability of fragments of the Azoarcus group I intron ribozyme to covalently self-assemble via genotype-selfish and genotype-cooperative interactions into full-length ribozymes to investigate the dynamics of small (three- and four-membered) networks. We focused on the influence of a three-membered core network on the incorporation of additional nodes, and on the degree and direction of connectivity as single new nodes are added to this core. We confirmed experimentally the predictions that additional links to a core should enhance overall network growth rates, but that the directionality of the link (a "giver" or a "receiver") impacts the growth of the core itself. Additionally, we used a simple mathematical model based on the first-order effects of lower-level interactions to predict the growth of more complex networks, and find that such a model can, to a first approximation, predict the ordinal rankings of nodes once a steady-state distribution has been reached.

Group I Intron Internal Guide Sequence Binding Strength as a Component of Ribozyme Network Formation.

Origins-of-life research requires searching for a plausible transition from simple chemicals to larger macromolecules that can both hold information and catalyze their own production. We have previously shown that some group I intron ribozymes possess the ability to help synthesize other ribozyme genotypes by recombination reactions in small networks in an autocatalytic fashion. By simplifying these recombination reactions, using fluorescent anisotropy, we quantified the thermodynamic binding strength between two nucleotides of two group I intron RNA fragments for all 16 possible genotype combinations. We provide evidence that the binding strength (KD) between the 3-nucleotide internal guide sequence (IGS) of one ribozyme and its complement in another is correlated to the catalytic ability of the ribozyme. This work demonstrates that one can begin to deconstruct the thermodynamic basis of information in prebiotic RNA systems.

Sex in a test tube: testing the benefits of in vitro recombination.

The origin and evolution of sex, and the associated role of recombination, present a major problem in biology. Sex typically involves recombination of closely related DNA or RNA sequences, which is fundamentally a random process that creates but also breaks up beneficial allele combinations. Directed evolution experiments, which combine in vitro mutation and recombination protocols with in vitro or in vivo selection, have proved to be an effective approach for improving functionality of nucleic acids and enzymes. As this approach allows extreme control over evolutionary conditions and parameters, it also facilitates the detection of small or position-specific recombination benefits and benefits associated with recombination between highly divergent genotypes. Yet, in vitro approaches have been largely exploratory and motivated by obtaining improved end products rather than testing hypotheses of recombination benefits. Here, we review the various experimental systems and approaches used by in vitro studies of recombination, discuss what they say about the evolutionary role of recombination, and sketch their potential for addressing extant questions about the evolutionary role of sex and recombination, in particular on complex fitness landscapes. We also review recent insights into the role of 'extracellular recombination' during the origin of life.This article is part of the themed issue 'Weird sex: the underappreciated diversity of sexual reproduction'.

Dynamics of prebiotic RNA reproduction illuminated by chemical game theory.

Many origins-of-life scenarios depict a situation in which there are common and potentially scarce resources needed by molecules that compete for survival and reproduction. The dynamics of RNA assembly in a complex mixture of sequences is a frequency-dependent process and mimics such scenarios. By synthesizing Azoarcus ribozyme genotypes that differ in their single-nucleotide interactions with other genotypes, we can create molecules that interact among each other to reproduce. Pairwise interplays between RNAs involve both cooperation and selfishness, quantifiable in a 2 × 2 payoff matrix. We show that a simple model of differential equations based on chemical kinetics accurately predicts the outcomes of these molecular competitions using simple rate inputs into these matrices. In some cases, we find that mixtures of different RNAs reproduce much better than each RNA type alone, reflecting a molecular form of reciprocal cooperation. We also demonstrate that three RNA genotypes can stably coexist in a rock-paper-scissors analog. Our experiments suggest a new type of evolutionary game dynamics, called prelife game dynamics or chemical game dynamics. These operate without template-directed replication, illustrating how small networks of RNAs could have developed and evolved in an RNA world.

The elusive quest for RNA knots.

Physical entanglement, and particularly knots arise spontaneously in equilibrated polymers that are sufficiently long and densely packed. Biopolymers are no exceptions: knots have long been known to occur in proteins as well as in encapsidated viral DNA. The rapidly growing number of RNA structures has recently made it possible to investigate the incidence of physical knots in this type of biomolecule, too. Strikingly, no knots have been found to date in the known RNA structures. In this Point of View Article we discuss the absence of knots in currently available RNAs and consider the reasons why knots in RNA have not yet been found, despite the expectation that they should exist in Nature. We conclude by singling out a number of RNA sequences that, based on the properties of their predicted secondary structures, are good candidates for knotted RNAs.

Prebiotic network evolution: six key parameters.

The origins of life likely required the cooperation among a set of molecular species interacting in a network. If so, then the earliest modes of evolutionary change would have been governed by the manners and mechanisms by which networks change their compositions over time. For molecular events, especially those in a pre-biological setting, these mechanisms have rarely been considered. We are only recently learning to apply the results of mathematical analyses of network dynamics to prebiotic events. Here, we attempt to forge connections between such analyses and the current state of knowledge in prebiotic chemistry. Of the many possible influences that could direct primordial network, six parameters emerge as the most influential when one considers the molecular characteristics of the best candidates for the emergence of biological information: polypeptides, RNA-like polymers, and lipids. These parameters are viable cores, connectivity kinetics, information control, scalability, resource availability, and compartmentalization. These parameters, both individually and jointly, guide the aggregate evolution of collectively autocatalytic sets. We are now in a position to translate these conclusions into a laboratory setting and test empirically the dynamics of prebiotic network evolution.

RNA-directed recombination of RNA in vitro.

Construction of long RNAs can be achieved in vitro by using ribozymes to recombine shorter RNAs. This can be a useful technique to prepare RNAs when the final product is either very long or contains chemical modifications that are difficult to incorporate using standard in vitro transcription techniques. Here, we describe the use of the Azoarcus group I intron ribozyme to recombine shorter RNAs into longer ones. This ribozyme is a generalized RNA recombinase ribozyme that operates rapidly and with high efficiency.

The RNA World: molecular cooperation at the origins of life.

The RNA World concept posits that there was a period of time in primitive Earth's history - about 4 billion years ago - when the primary living substance was RNA or something chemically similar. In the past 50 years, this idea has gone from speculation to a prevailing idea. In this Review, we summarize the key logic behind the RNA World and describe some of the most important recent advances that have been made to support and expand this logic. We also discuss the ways in which molecular cooperation involving RNAs would facilitate the emergence and early evolution of life. The immediate future of RNA World research should be a very dynamic one.

The RNA World: 4,000,000,050 years old.

The RNA World is now some four billion years behind us, but only recently turned 50 as a human hypothesis. [...].

Empirical demonstration of environmental sensing in catalytic RNA: evolution of interpretive behavior at the origins of life.

BACKGROUND: The origins of life on the Earth required chemical entities to interact with their environments in ways that could respond to natural selection. The concept of interpretation, where biotic entities use signs in their environment as proxy for the existence of other items of selective value in their environment, has been proposed on theoretical grounds to be relevant to the origins and early evolution of life. However this concept has not been demonstrated empirically. RESULTS: Here, we present data that certain catalytic RNA sequences have properties that would enable interpretation of divalent cation levels in their environment. By assaying the responsiveness of two variants of the Tetrahymena ribozyme to the Ca(2+) ion as a sign for the more catalytically useful Mg(2+) ion, we show an empirical proof-of-principle that interpretation can be an evolvable trait in RNA, often suggested as a model system for early life. In particular we demonstrate that in vitro, the wild-type version of the Tetrahymena ribozyme is not interpretive, in that it cannot use Ca(2+) as a sign for Mg(2+). Yet a variant of this sequence containing five mutations that alter its ability to utilize the Ca(2+) ion engenders a strong interpretive characteristic in this RNA. CONCLUSIONS: We have shown that RNA molecules in a test tube can meet the minimum criteria for the evolution of interpretive behaviour in regards to their responses to divalent metal ion concentrations in their environment. Interpretation in RNA molecules provides a property entirely dependent on natural physico-chemical interactions, but capable of shaping the evolutionary trajectory of macromolecules, especially in the earliest stages of life's history.