We report the first example of RNA labeling based on inverse electron-demand Diels-Alder reactions. Both chemically synthesized and enzymatically transcribed RNAs were successfully modified with biotin or a fluorescent label. This approach works efficiently under mild conditions in water and does not require transition metals.

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.

33Evolution via template-based replication 1-4 was probably preceded by a more rudimentary form 34 of evolution based on networks of autocatalytic reactions [5][6][7][8] . However, reaction networks 35 possessing the Darwinian properties of variation (in composition, the catalysts present and their 36 relative amounts), differential reproduction (accumulation of products), and heredity 37 (persistence of composition), have so far not been identified. Here we show that networks of 38 catalytic RNAs can possess certain properties of Darwinian systems, that these properties are 39 controlled by network topology, and characterize important trade-offs between them. By 40 combining barcoded sequencing with droplet microfluidics, we screened ̴ 20,000 reactions 41 corresponding to more than 1,800 distinct networks of ribozymes that catalyse their own 42 formation from RNA fragments. We found that more highly connected networks tend to 43 reproduce more quickly (accumulate more ribozymes) and be more robust to perturbations, 44 indicating a trade-off between variation and reproduction. Variations are strongest when adding 45 upstream ribozymes with novel reaction specificities (innovations) which target weakly 46 connected networks. In turn, innovations increase connectivity, thus buffer against further 47 Main 59In the prebiotic world, the spontaneous appearance of an RNA polymerase ribozyme (a 60 replicase) with sufficient processivity to allow self-replication and enough fidelity to avoid an 61 error catastrophe 5,6 seems unlikely, given that known replicases are long (>165 nt) and 62 structurally complex 1-4 . However, theoretical studies suggest that earlier modes of evolution 63 could have been supported by autocatalytic sets, where reproduction results from networks of 64 more rudimentary catalysts [11][12][13] . Consistently, recent experiments indicate that RNA 65 polymerase ribozymes can assemble from catalytic networks of RNA oligomers 14 , suggesting 66 that replicases may have emerged as components of such networks. Nevertheless, sustaining 67 evolution in reaction networks is by no means trivial, as the Darwinian properties of variation, 68 heredity and selection are mediated by chemical compositions (the proportion of different 69 chemical species) rather than the copying of a sequence. Furthermore, the possibility to evolve 70 chemistries may be constained by trade-offs between these properties. For instance, robustness 71 to environmental perturbations and persistence of compositions are necessary for selection to 72 act, but must be balanced with variation to explore novel states. So far, none of these Darwinian 73properties, nor their interplay, have been experimentally studied at a large scale in a 74 prebiotically relevant system. 75We studied an experimental model of autocatalytic RNAs derived from the group I intron of 76 the Azoarcus bacterium 15 . Fragments (denoted WXY and Z) assemble into non-covalent 77 complexes that catalyze the formation of more efficient covalent ribozymes 16,17 (denoted 78...

Catalytic RNAs are attractive objects for studying molecular evolution. To understand how RNA libraries can evolve from randomness toward highly active catalysts, we analyze the original samples that led to the discovery of Diels–Alderase ribozymes by next-generation sequencing. Known structure-activity relationships are used to correlate abundance with catalytic performance. We find that efficient catalysts arose not just from selection for reactivity among the members of the starting library, but from improvement of less potent precursors by mutations. We observe changes in the ribozyme population in response to increasing selection pressure. Surprisingly, even after many rounds of enrichment, the libraries are highly diverse, suggesting that potential catalysts are more abundant in random space than generally thought. To highlight the use of next-generation sequencing as a tool for in vitro selections, we also apply this technique to a recent, less characterized ribozyme selection. Making use of the correlation between sequence evolution and catalytic activity, we predict mutations that improve ribozyme activity and validate them biochemically. Our study reveals principles underlying ribozyme in vitro selections and provides guidelines to render future selections more efficient, as well as to predict the conservation of key structural elements, allowing the rational improvement of catalysts.

Understanding the emergence of life from (primitive) abiotic components has arguably been one of the deepest and yet one of the most elusive scientific questions. Notwithstanding the lack of a clear definition for a living system, it is widely argued that heredity (involving self-reproduction) along with compartmentalization and metabolism are key features that contrast living systems from their non-living counterparts. A minimal living system may be viewed as “a self-sustaining chemical system capable of Darwinian evolution”. It has been proposed that autocatalytic sets of chemical reactions (ACSs) could serve as a mechanism to establish chemical compositional identity, heritable self-reproduction, and evolution in a minimal chemical system. Following years of theoretical work, autocatalytic chemical systems have been constructed experimentally using a wide variety of substrates, and most studies, thus far, have focused on the demonstration of chemical self-reproduction under specific conditions. While several recent experimental studies have raised the possibility of carrying out some aspects of experimental evolution using autocatalytic reaction networks, there remain many open challenges. In this review, we start by evaluating theoretical studies of ACSs specifically with a view to establish the conditions required for such chemical systems to exhibit self-reproduction and Darwinian evolution. Then, we follow with an extensive overview of experimental ACS systems and use the theoretically established conditions to critically evaluate these empirical systems for their potential to exhibit Darwinian evolution. We identify various technical and conceptual challenges limiting experimental progress and, finally, conclude with some remarks about open questions.

The enzymatic catalysis of difficult chemical reactions often requires the utilization of mechanisms completely different from those used in the uncatalyzed reaction. The catalytic triad of the serine proteases is an illustrative example for the cooperation of functional groups to achieve the hydrolysis of a very stable peptide bond via a covalent intermediate. Ribozymes for this demanding reaction that use similar mechanisms have neither been discovered nor developed to date. Here, we challenge a combinatorial RNA library with an active site-directed mechanistic inhibitor of serine proteases in order to identify RNA molecules with a chemical reactivity comparable to the residues in the catalytic center of thrombin. The adduct formed by this inhibitor is thought to mimic the first transition state in a complex reaction pathway and contains a weak electrophile. Indeed, various RNAs are enriched that accelerate their covalent attachment to the inhibitor, and several of them share a common motif that features a four-way junction. These RNAs specifically alkylate the N7-position of one particular guanosine, precisely recognizing structural features of the inhibitor. The selected RNAs may represent a valuable starting point for the further evolution of ribozymes with protease activity.

Here we report an efficient method for the synthesis of RNA-peptide conjugates by inverse-electron demand Diels-Alder reaction. Various dienophiles were enzymatically incorporated into RNA and reacted with a chemically synthesized diene-modified peptide. The Diels-Alder reaction proceeds with near-quantitative yields in aqueous solution with stoichiometric amounts of reactants, even at low micromolar concentrations.

We demonstrate that a recombinase ribozyme achieves multiple functions in the same reaction network: self-reproduction, iterative elongation and circularization of other RNAs, leading to synthesis of diverse products predicted by...