RNA World Research Articles

Biomathematical enzyme kinetics model of prebiotic autocatalytic RNA networks: degenerating parasite-specific hyperparasite catalysts confer parasite resistance and herald the birth of molecular immunity.

Catalysis and specifically autocatalysis are the quintessential building blocks of life. Yet, although autocatalytic networks are necessary, they are not sufficient for the emergence of life-like properties, such as replication and adaptation. The ultimate and potentially fatal threat faced by molecular replicators is parasitism; if the polymerase error rate exceeds a critical threshold, even the fittest molecular species will disappear. Here we have developed an autocatalytic RNA early life mathematical network model based on enzyme kinetics, specifically the steady-state approximation. We confirm previous models showing that these second-order autocatalytic cycles are sustainable, provided there is a sufficient nucleotide pool. However, molecular parasites become untenable unless they sequentially degenerate to hyperparasites (i.e. parasites of parasites). Parasite resistance-a parasite-specific host response decreasing parasite fitness-is acquired gradually, and eventually involves an increased binding affinity of hyperparasites for parasites. Our model is supported at three levels; firstly, ribozyme polymerases display Michaelis-Menten saturation kinetics and comply with the steady-state approximation. Secondly, ribozyme polymerases are capable of sustainable auto-amplification and of surmounting the fatal error threshold. Thirdly, with growing sequence divergence of host and parasite catalysts, the probability of self-binding is expected to increase and the trend towards cross-reactivity to diminish. Our model predicts that primordial host-RNA populations evolved via an arms race towards a host-parasite-hyperparasite catalyst trio that conferred parasite resistance within an RNA replicator niche. While molecular parasites have traditionally been viewed as a nuisance, our model argues for their integration into the host habitat rather than their separation. It adds another mechanism-with biochemical precision-by which parasitism can be tamed and offers an attractive explanation for the universal coexistence of catalyst trios within prokaryotes and the virosphere, heralding the birth of a primitive molecular immunity.

Prebiotic RNA self-assembling and the origin of life: Mechanistic and molecular modeling rationale for explaining the prebiotic origin and replication of RNA.

In recent years, a great interest has been focused on the prebiotic origin of nucleic acids and life on Earth. An attractive idea is that life was initially based on an autocatalytic and autoreplicative RNA (the RNA-world). RNA duplexes are right-handed helical chains with antiparallel orientation, but the rationale for these features is not yet known. An antiparallel (inverted) stacking of purine nucleosides was reported in crystallographic studies. Molecular modeling also supports the inverted orientation of nucleosides. This preferential stacking can also appear when nucleosides are included in a montmorillonite clay matrix. Free-energy values and geometrical parameters show that D-ribose chirality is preferred for the formation of right-handed RNA molecules. Thus, a "zipper" model with antiparallel and auto-intercalated nucleosides linked by phosphate groups can be proposed to form single RNA chains. Unstacking with strand separation and base pairing by H-bonding, results in shortening and inclination of ribose-phosphate chains, leading to right-handed helicity and antiparallel duplexes. Incorporation of complementary precursors on the major groove template by a self-assembly mechanism provides a prebiotic (non-enzymatic) "tetris" replication model by formation of a transient RNA tetrad and tetraplex. Original hairpin motifs appear as simple building units that form typical RNA structures such as hammerheads, cloverleaves and dumbbells. They occur today in the circular viroids and virusoids, as well as in highly branched and complex rRNA molecules.

Activation of Caged Functional RNAs by An Oxidative Transformation.

RNA exhibits remarkable capacity as a functional polymer, with broader catalytic and ligand-binding capability than previously thought. Despite this, the low side chain diversity present in nucleic acids (two purines and two pyrimidines) relative to proteins (20+ side chains of varied charge, polarity, and chemical functionality) limits the capacity of functional RNAs to act as environmentally responsive polymers, as is possible for peptide-based receptors and catalysts. Here we show that incorporation of the modified nucleobase 2-thiouridine (2sU) into functional (aptamer and ribozyme) RNAs produces functionally inactivated polymers that can be activated by oxidative treatment. 2-thiouridine lacksthe 2-position oxygen found in uridine, altering its hydrogen bonding pattern. This limits critical interactions (e. g., G-U wobble pairs) that allow for proper folding. Oxidative desulfurization of the incorporated 2-thiouridine moieties to uridine relieves this inability to fold properly, enabling recovery of function. This demonstration of expanded roles for RNA as environmentally responsive functional polymers challenges the notion that they are not known to be redox-sensitive. Harnessing redox switchability in RNA could regulate cellular activities such as translation, or allow switching RNA between a "template" and a "catalytic" state in "RNA World" scenarios or in synthetic biology.

Diversity and evolution of viroids and viroid-like agents with circular RNA genomes revealed by metatranscriptome mining.

Viroids, the agents of several plant diseases, are the smallest and simplest known replicators that consist of covalently closed circular (ccc) RNA molecules between 200 and 400 nucleotides in size. Viroids encode no proteins and rely on host RNA polymerases for replication, but some contain ribozymes involved in replication intermediate processing. Although other viroid-like agents with cccRNAs genomes, such as satellite RNAs, ribozyviruses and retrozymes, have been discovered, until recently, the spread of these agents in the biosphere appeared narrow, and their actual diversity and evolution remained poorly understood. Extensive, targeted metatranscriptome mining dramatically expanded the known diversity of cccRNAs genomes. These searches identified numerous, diverse viroid-like cccRNAs, many found in environments devoid of plant and animal material, suggesting replication in unicellular eukaryotic and/or prokaryotic hosts. Several cccRNAs are targeted by CRISPR systems, supporting their association with bacteria. In addition to small cccRNAs in the viroid size range, a broad variety of ribozyviruses and novel viruses with cccRNAs genomes, with genomes reaching nearly 5 kilobases, were discovered. Thus, metatranscriptome mining shows that the diversity of viroid-like cccRNAs genomes is far greater than previously suspected, prompting reassessment of the relevance of these replicators for understanding the primordial RNA world.

Indirect Formation of Peptide Bonds as a Prelude to Ribosomal Transpeptidation.

The catalytic competency of the ribosome in extant protein biosynthesis is thought to arise primarily from two sources: an ability to precisely juxtapose the termini of two key substrates─3'-aminoacyl and N-acyl-aminoacyl tRNAs─and an ability to ease direct transpeptidation by their desolvation and encapsulation. In the absence of ribosomal, or enzymatic, protection, however, these activated alkyl esters undergo efficient hydrolysis, while significant entropic barriers serve to hamper their intermolecular cross-aminolysis in bulk water. Given that the spontaneous emergence of a catalyst of comparable size and sophistication to the ribosome in a prebiotic RNA world would appear implausible, it is thus natural to ask how appreciable peptide formation could have occurred with such substrates in bulk water without the aid of advanced ribozymatic catalysis. Using a combination of fluorine-tagged aminoacyl adenylate esters, in situ monitoring by 19F{1H} NMR spectroscopy, analytical deconvolution of kinetics, pH-rate profile analysis, and temperature-dependence studies, we here explore the mechanistic landscape of indirect amidation, via transesterification and O-to-N rearrangement, as a highly efficient, alternative manifold for transpeptidation that may have served as a prelude to ribosomal peptide synthesis. Our results suggest a potentially overlooked role for those amino acids implicated by the cyanosulfidic reaction network with hydroxyl side chains (Ser and Thr), and they also help to resolve some outstanding ambiguities in the broader literature regarding studies of similar systems (e.g., aminolyzes with Tris buffer). The evolutionary implications of this mode of peptide synthesis and the involvement of a very specific subset of amino acids are discussed.

Origin of Life: A Symmetry-Breaking Physical Phase Transition

The origin of life has previously been subject to numerous studies and hypotheses. Typically, related models focus on the emergence of chemical networks such as the RNA world or the Krebs energy cycle. Here, the onset of life is described as a symmetry-breaking kinetic phase transition. The novel symmetry of life is the arbitrariness of code that is fundamental to symbolic information processing, coining all forms of life from the very beginning. Symbols evolved from non-symbolic, structural information of the inanimate physical world. The responsible transition process was discovered a century ago in behavioural biology, regarded as ‘ritualisation’. The physical properties of this transition include neutral Lyapunov stability and critical fluctuations in the associated Goldstone modes. As a conceptual model, a hypothetical simple molecular ritualisation process is suggested, along with the emergent semiotics of symbolic information processing.

RNA World with Inhibitors.

During the evolution of the RNA World, compartments, which were fragments of space surrounded by a primitive lipid membrane, had to have emerged. These led eventually to the formation of modern cellular membranes. Inside these compartments, another process had to take place-switching from RNA to DNA as a primary storage of genetic information. The latter part needed a handful of enzymes for the DNA to be able to perform its function. A natural question arises, i.e., how the concentration of all vital molecules could have been kept in check without modern cellular mechanisms. The authors propose a theory on how it could have worked during early stages, using only short RNA molecules, which could have emerged spontaneously. The hypothesis was analysed mathematically and tested against different scenarios by using computer simulations.

Intrinsically disordered RNA-binding motifs cooperate to catalyze RNA folding and drive phase separation.

RNA-binding proteins are essential for gene regulation and the spatial organization of cells. Here, we report that the yeast ribosome biogenesis factor Loc1p is an intrinsically disordered RNA-binding protein with eight repeating positively charged, unstructured nucleic acid binding (PUN) motifs. While a single of these previously undefined motifs stabilizes folded RNAs, multiple copies strongly cooperate to catalyze RNA folding. In the presence of RNA, these multivalent PUN motifs drive phase separation. Proteome-wide searches in pro- and eukaryotes for proteins with similar arrays of PUN motifs reveal a strong enrichment in RNA-mediated processes and DNA remodeling. Thus, PUN motifs are potentially involved in a large variety of RNA- and DNA-related processes by concentrating them in membraneless organelles. The general function and wide distribution of PUN motifs across species suggest that in an ancient 'RNA world' PUN-like motifs may have supported the correct folding of early ribozymes.

Overcoming nucleotide bias in the nonenzymatic copying of RNA templates.

The RNA World hypothesis posits that RNA was the molecule of both heredity and function during the emergence of life. This hypothesis implies that RNA templates can be copied, and ultimately replicated, without the catalytic aid of evolved enzymes. A major problem with nonenzymatic template-directed polymerization has been the very poor copying of sequences containing rA and rU. Here, we overcome that problem by using a prebiotically plausible mixture of RNA mononucleotides and random-sequence oligonucleotides, all activated by methyl isocyanide chemistry, that direct the uniform copying of arbitrary-sequence templates, including those harboring rA and rU. We further show that the use of this mixture in copying reactions suppresses copying errors while also generating a more uniform distribution of mismatches than observed for simpler systems. We find that oligonucleotide competition for template binding sites, oligonucleotide ligation and the template binding properties of reactant intermediates work together to reduce product sequence bias and errors. Finally, we show that iterative cycling of templated polymerization and activation chemistry improves the yields of random-sequence products. These results for random-sequence template copying are a significant advance in the pursuit of nonenzymatic RNA replication.

Directionality theory and the origin of life.

The origin of cellular life can be described in terms of the transition from inorganic matter to the emergence of cooperative assemblies of organic matter: DNA and proteins, capable of replication and metabolism. Directionality theory is a mathematical theory of the collective behaviour of networks of organic matter: activated macromolecules, cells and higher organisms. Evolutionary entropy, a generalization of the thermodynamic entropy of Boltzmann, is a statistical measure of the cooperativity of the biotic components. The cornerstone of Directionality theory is the Entropic Principle of Evolution: evolutionary entropy increases in systems driven by a stable energy source, and decreases in systems subject to a fluctuating energy source. This article invokes the Entropic Principle of Evolution-an extension to biological systems of the Second Law of Thermodynamics-to provide an adaptive rationale for the following sequence of transformations that define the emergence of cellular life: (i) the self-assembly of activated macromolecules from inorganic matter; (ii) the emergence of an RNA world, defined by RNA molecules with catalytic and replicative properties; and (iii) the origin of cellular life, the integration of the three carbon-based polymers-DNA, proteins and lipids, to generate a metabolic and replicative unit.

A Compendium of G-Flipon Biological Functions That Have Experimental Validation.

As with all new fields of discovery, work on the biological role of G-quadruplexes (GQs) has produced a number of results that at first glance are quite baffling, sometimes because they do not fit well together, but mostly because they are different from commonly held expectations. Like other classes of flipons, those that form G-quadruplexes have a repeat sequence motif that enables the fold. The canonical DNA motif (G3N1-7)3G3, where N is any nucleotide and G is guanine, is a feature that is under active selection in avian and mammalian genomes. The involvement of G-flipons in genome maintenance traces back to the invertebrate Caenorhabditis elegans and to ancient DNA repair pathways. The role of GQs in transcription is supported by the observation that yeast Rap1 protein binds both B-DNA, in a sequence-specific manner, and GQs, in a structure-specific manner, through the same helix. Other sequence-specific transcription factors (TFs) also engage both conformations to actuate cellular transactions. Noncoding RNAs can also modulate GQ formation in a sequence-specific manner and engage the same cellular machinery as localized by TFs, linking the ancient RNA world with the modern protein world. The coevolution of noncoding RNAs and sequence-specific proteins is supported by studies of early embryonic development, where the transient formation of G-quadruplexes coordinates the epigenetic specification of cell fate.

Life should be redefined: Any molecule with the ability to self-replicate should be considered life.

Understanding the nature of life and its propensity for reproduction has long been a question that humans aspire to answer. Reproduction, a defining characteristic of life, fundamentally involves the replication of genetic material, be it DNA or RNA. The driving force behind this replication process has always intrigued scientists. In recent years, theories involving selfish genes, the RNA world, and entropic forces have been proposed by some scholars. These theories seem to suggest that life, as we know it, exists solely in Earth's environment and is based on a single type of genetic material, either DNA or RNA. However, if we broaden our definition of life to include any replicable molecules, we might be able to transcend traditional thought. This could potentially enhance our understanding of the impetus behind DNA replication and provide deeper insights into the essence of life.

Prebiotic chiral transfer from self-aminoacylating ribozymes may favor either handedness

Modern life is essentially homochiral, containing D-sugars in nucleic acid backbones and L-amino acids in proteins. Since coded proteins are theorized to have developed from a prebiotic RNA World, the homochirality of L-amino acids observed in all known life presumably resulted from chiral transfer from a homochiral D-RNA World. This transfer would have been mediated by aminoacyl-RNAs defining the genetic code. Previous work on aminoacyl transfer using tRNA mimics has suggested that aminoacylation using D-RNA may be inherently biased toward reactivity with L-amino acids, implying a deterministic path from a D-RNA World to L-proteins. Using a model system of self-aminoacylating D-ribozymes and epimerizable activated amino acid analogs, we test the chiral selectivity of 15 ribozymes derived from an exhaustive search of sequence space. All of the ribozymes exhibit detectable selectivity, and a substantial fraction react preferentially to produce the D-enantiomer of the product. Furthermore, chiral preference is conserved within sequence families. These results are consistent with the transfer of chiral information from RNA to proteins but do not support an intrinsic bias of D-RNA for L-amino acids. Different aminoacylation structures result in different directions of chiral selectivity, such that L-proteins need not emerge from a D-RNA World.

A potential role for RNA aminoacylation prior to its role in peptide synthesis

Coded ribosomal peptide synthesis could not have evolved unless its sequence and amino acid-specific aminoacylated tRNA substrates already existed. We therefore wondered whether aminoacylated RNAs might have served some primordial function prior to their role in protein synthesis. Here, we show that specific RNA sequences can be nonenzymatically aminoacylated and ligated to produce amino acid-bridged stem-loop RNAs. We used deep sequencing to identify RNAs that undergo highly efficient glycine aminoacylation followed by loop-closing ligation. The crystal structure of one such glycine-bridged RNA hairpin reveals a compact internally stabilized structure with the same eponymous T-loop architecture that is found in many noncoding RNAs, including the modern tRNA. We demonstrate that the T-loop-assisted amino acid bridging of RNA oligonucleotides enables the rapid template-free assembly of a chimeric version of an aminoacyl-RNA synthetase ribozyme. We suggest that the primordial assembly of amino acid-bridged chimeric ribozymes provides a direct and facile route for the covalent incorporation of amino acids into RNA. A greater functionality of covalently incorporated amino acids could contribute to enhanced ribozyme catalysis, providing a driving force for the evolution of sequence and amino acid-specific aminoacyl-RNA synthetase ribozymes in the RNA World. The synthesis of specifically aminoacylated RNAs, an unlikely prospect for nonenzymatic reactions but a likely one for ribozymes, could have set the stage for the subsequent evolution of coded protein synthesis.

An ontology-based knowledge graph for representing interactions involving RNA molecules

The “RNA world” represents a novel frontier for the study of fundamental biological processes and human diseases and is paving the way for the development of new drugs tailored to each patient’s biomolecular characteristics. Although scientific data about coding and non-coding RNA molecules are constantly produced and available from public repositories, they are scattered across different databases and a centralized, uniform, and semantically consistent representation of the “RNA world” is still lacking. We propose RNA-KG, a knowledge graph (KG) encompassing biological knowledge about RNAs gathered from more than 60 public databases, integrating functional relationships with genes, proteins, and chemicals and ontologically grounded biomedical concepts. To develop RNA-KG, we first identified, pre-processed, and characterized each data source; next, we built a meta-graph that provides an ontological description of the KG by representing all the bio-molecular entities and medical concepts of interest in this domain, as well as the types of interactions connecting them. Finally, we leveraged an instance-based semantically abstracted knowledge model to specify the ontological alignment according to which RNA-KG was generated. RNA-KG can be downloaded in different formats and also queried by a SPARQL endpoint. A thorough topological analysis of the resulting heterogeneous graph provides further insights into the characteristics of the “RNA world”. RNA-KG can be both directly explored and visualized, and/or analyzed by applying computational methods to infer bio-medical knowledge from its heterogeneous nodes and edges. The resource can be easily updated with new experimental data, and specific views of the overall KG can be extracted according to the bio-medical problem to be studied.

The Last Universal Common Ancestor of Ribosome-Encoding Organisms: Portrait of LUCA.

The existence of LUCA in the distant past is the logical consequence of the binary mechanism of cell division. The biosphere in which LUCA and contemporaries were living was the product of a long cellular evolution from the origin of life to the second age of the RNA world. A parsimonious scenario suggests that the molecular fabric of LUCA was much simpler than those of modern organisms, explaining why the evolutionary tempo was faster at the time of LUCA than it was during the diversification of the three domains. Although LUCA was possibly equipped with a RNA genome and most likely lacked an ATP synthase, it was already able to perform basic metabolic functions and to produce efficient proteins. However, the proteome of LUCA and its inferred metabolism remains to be correctly explored by in-depth phylogenomic analyses and updated datasets. LUCA was probably a mesophile or a moderate thermophile since phylogenetic analyses indicate that it lacked reverse gyrase, an enzyme systematically present in all hyperthermophiles. The debate about the position of Eukarya in the tree of life, either sister group to Archaea or descendants of Archaea, has important implications to draw the portrait of LUCA. In the second alternative, one can a priori exclude the presence of specific eukaryotic features in LUCA. In contrast, if Archaea and Eukarya are sister group, some eukaryotic features, such as the spliceosome, might have been present in LUCA and later lost in Archaea and Bacteria. The nature of the LUCA virome is another matter of debate. I suggest here that DNA viruses only originated during the diversification of the three domains from an RNA-based LUCA to explain the odd distribution pattern of DNA viruses in the tree of life.

Protein Fold Usages in Ribosomes: Another Glance to the Past.

The analysis of protein fold usage, similar to codon usage, offers profound insights into the evolution of biological systems and the origins of modern proteomes. While previous studies have examined fold distribution in modern genomes, our study focuses on the comparative distribution and usage of protein folds in ribosomes across bacteria, archaea, and eukaryotes. We identify the prevalence of certain 'super-ribosome folds,' such as the OB fold in bacteria and the SH3 domain in archaea and eukaryotes. The observed protein fold distribution in the ribosomes announces the future power-law distribution where only a few folds are highly prevalent, and most are rare. Additionally, we highlight the presence of three copies of proto-Rossmann folds in ribosomes across all kingdoms, showing its ancient and fundamental role in ribosomal structure and function. Our study also explores early mechanisms of molecular convergence, where different protein folds bind equivalent ribosomal RNA structures in ribosomes across different kingdoms. This comparative analysis enhances our understanding of ribosomal evolution, particularly the distinct evolutionary paths of the large and small subunits, and underscores the complex interplay between RNA and protein components in the transition from the RNA world to modern cellular life. Transcending the concept of folds also makes it possible to group a large number of ribosomal proteins into five categories of urfolds or metafolds, which could attest to their ancestral character and common origins. This work also demonstrates that the gradual acquisition of extensions by simple but ordered folds constitutes an inexorable evolutionary mechanism. This observation supports the idea that simple but structured ribosomal proteins preceded the development of their disordered extensions.

Nicotinamide Riboside: What It Takes to Incorporate It into RNA.

Nicotinamide is an important functional compound and, in the form of nicotinamide adenine dinucleotide (NAD), is used as a co-factor by protein-based enzymes to catalyze redox reactions. In the context of the RNA world hypothesis, it is therefore reasonable to assume that ancestral ribozymes could have used co-factors such as NAD or its simpler analog nicotinamide riboside (NAR) to catalyze redox reactions. The only described example of such an engineered ribozyme uses a nicotinamide moiety bound to the ribozyme through non-covalent interactions. Covalent attachment of NAR to RNA could be advantageous, but the demonstration of such scenarios to date has suffered from the chemical instability of both NAR and its reduced form, NARH, making their use in oligonucleotide synthesis less straightforward. Here, we review the literature describing the chemical properties of the oxidized and reduced species of NAR, their synthesis, and previous attempts to incorporate either species into RNA. We discuss how to overcome the stability problem and succeed in generating RNA structures incorporating NAR.

Circular at the very beginning: on the initial genomes in the RNA world

ABSTRACT It is likely that an RNA world existed in early life, when RNA played both the roles of the genome and functional molecules, thereby undergoing Darwinian evolution. However, even with only one type of polymer, it seems quite necessary to introduce a labour division concerning these two roles because folding is required for functional molecules (ribozymes) but unfavourable for the genome (as a template in replication). Notably, while ribozymes tend to have adopted a linear form for folding without constraints, a circular form, which might have been topologically hindered in folding, seems more suitable for an RNA template. Another advantage of involving a circular genome could have been to resist RNA’s end-degradation. Here, we explore the scenario of a circular RNA genome plus linear ribozyme(s) at the precellular stage of the RNA world through computer modelling. The results suggest that a one-gene scene could have been ‘maintained’, albeit with rather a low efficiency for the circular genome to produce the ribozyme, which required precise chain-break or chain-synthesis. This strict requirement may have been relieved by introducing a ‘noncoding’ sequence into the genome, which had the potential to derive a second gene through mutation. A two-gene scene may have ‘run well’ with the two corresponding ribozymes promoting the replication of the circular genome from different respects. Circular genomes with more genes might have arisen later in RNA-based protocells. Therefore, circular genomes, which are common in the modern living world, may have had their ‘root’ at the very beginning of life.

Protocell Effects on RNA Folding, Function, and Evolution.

ConspectusCreating a living system from nonliving matter is a great challenge in chemistry and biophysics. The early history of life can provide inspiration from the idea of the prebiotic "RNA World" established by ribozymes, in which all genetic and catalytic activities were executed by RNA. Such a system could be much simpler than the interdependent central dogma characterizing life today. At the same time, cooperative systems require a mechanism such as cellular compartmentalization in order to survive and evolve. Minimal cells might therefore consist of simple vesicles enclosing a prebiotic RNA metabolism.The internal volume of a vesicle is a distinctive environment due to its closed boundary, which alters diffusion and available volume for macromolecules and changes effective molecular concentrations, among other considerations. These physical effects are mechanistically distinct from chemical interactions, such as electrostatic repulsion, that might also occur between the membrane boundary and encapsulated contents. Both indirect and direct interactions between the membrane and RNA can give rise to nonintuitive, "emergent" behaviors in the model protocell system. We have been examining how encapsulation inside membrane vesicles would affect the folding and activity of entrapped RNA.Using biophysical techniques such as FRET, we characterized ribozyme folding and activity inside vesicles. Encapsulation inside model protocells generally promoted RNA folding, consistent with an excluded volume effect, independently of chemical interactions. This energetic stabilization translated into increased ribozyme activity in two different systems that were studied (hairpin ribozyme and self-aminoacylating RNAs). A particularly intriguing finding was that encapsulation could rescue the activity of mutant ribozymes, suggesting that encapsulation could affect not only folding and activity but also evolution. To study this further, we developed a high-throughput sequencing assay to measure the aminoacylation kinetics of many thousands of ribozyme variants in parallel. The results revealed an unexpected tendency for encapsulation to improve the better ribozyme variants more than worse variants. During evolution, this effect would create a tilted playing field, so to speak, that would give additional fitness gains to already-high-activity variants. According to Fisher's Fundamental Theorem of Natural Selection, the increased variance in fitness should manifest as faster evolutionary adaptation. This prediction was borne out experimentally during in vitro evolution, where we observed that the initially diverse ribozyme population converged more quickly to the most active sequences when they were encapsulated inside vesicles.The studies in this Account have expanded our understanding of emergent protocell behavior, by showing how simply entrapping an RNA inside a vesicle, which could occur spontaneously during vesicle formation, might profoundly affect the evolutionary landscape of the RNA. Because of the exponential dynamics of replication and selection, even small changes to activity and function could lead to major evolutionary consequences. By closely studying the details of minimal yet surprisingly complex protocells, we might one day trace a pathway from encapsulated RNA to a living system.