Molecular evolution can be conceptualized as a walk over a “fitness landscape”, or the function of fitness (e.g., catalytic activity) over the space of all possible sequences. Understanding evolution requires knowing the structure of the fitness landscape and identifying the viable evolutionary pathways through the landscape. However, the fitness landscape for any catalytic biomolecule is largely unknown. The evolution of catalytic RNA is of special interest because RNA is believed to have been foundational to early life. In particular, an essential activity leading to the genetic code would be the reaction of ribozymes with activated amino acids, such as 5(4H)-oxazolones, to form aminoacyl-RNA. Here we combine in vitro selection with a massively parallel kinetic assay to map a fitness landscape for self-aminoacylating RNA, with nearly complete coverage of sequence space in a central 21-nucleotide region. The method (SCAPE: sequencing to measure catalytic activity paired with in vitro evolution) shows that the landscape contains three major ribozyme families (landscape peaks). An analysis of evolutionary pathways shows that, while local optimization within a ribozyme family would be possible, optimization of activity over the entire landscape would be frustrated by large valleys of low activity. The sequence motifs associated with each peak represent different solutions to the problem of catalysis, so the inability to traverse the landscape globally corresponds to an inability to restructure the ribozyme without losing activity. The frustrated nature of the evolutionary network suggests that chance emergence of a ribozyme motif would be more important than optimization by natural selection.

Understanding how life arose is a fundamental problem of biology. Much progress has been made by adopting a synthetic and mechanistic perspective on originating life. We present a current view of the biochemistry of the origin of life, focusing on issues surrounding the emergence of an RNA World in which RNA dominated informational and functional roles. There is cause for optimism on this difficult problem: the prebiotic chemical inventory may not have been as nightmarishly complex as previously thought; the catalytic repertoire of ribozymes continues to expand, approaching the goal of self-replicating RNA; encapsulation in protocells provides evolutionary and biophysical advantages. Nevertheless, major issues remain unsolved, such as the origin of a genetic code. Attention to this field is particularly timely given the accelerating discovery and characterization of exoplanets.

Simulations of a chemical kinetics model, based on the free-energy relationships of classical primary nucleation theory, show that the deracemization phenomenon in systems of achiral or fast racemizing compounds yielding enantiopure crystals as the more stable solid phase is a true spontaneous mirror symmetry breaking process (SMSB). That is, the achievement of a stationary chiral state is more stable than the racemic one. The model translates the free-energy relationships determined by the existence of a critical size cluster to a chemical kinetics model, in which the consideration of forward and backward reaction rate constants avoids the misuse of network parameters that violate thermodynamic constraints (microreversibility principle), which would lead to apparent in silico SMSB. The model provides qualitative agreement for deracemizations by mechanical attrition of visible crystals, as well as for those obtained under temperature gradients. The analysis of the effect of the system parameters to obtain a SMSB scenario shows that the network possesses the principal characteristics of SMSB networks: 1) an enantioselective autocatalytic stage, corresponding to the non-linear kinetics of enantioselective (homochiral) cluster-to-cluster growth, and 2) the mutual inhibition step originating in the backward flow of chiral clusters towards smaller achiral clusters, or even to a racemizing monomer. The application of such a SMSB kinetic model to enantioselective polymerizations and to chiral biopolymers is discussed.

A central difficulty facing study of the origin of life on Earth is evaluating the relevance of different proposed prebiotic scenarios. Perhaps the most established feature of the origin of life was the progression through an RNA World, a prebiotic stage dominated by functional RNA. We use the appearance of proteins in the RNA World to understand the prebiotic milieu and develop a criterion to evaluate proposed synthetic scenarios. Current consensus suggests that the earliest amino acids of the genetic code were anionic or small hydrophobic or polar amino acids. However, the ability to interact with the RNA World would have been a crucial feature of early proteins. To determine which amino acids would be important for the RNA World, we analyze non-biological protein-aptamer complexes in which the RNA or DNA is the result of in vitro evolution. This approach avoids confounding effects of biological context and evolutionary history. We use bioinformatic analysis and molecular dynamics simulations to characterize these complexes. We find that positively charged and aromatic amino acids are over-represented whereas small hydrophobic amino acids are under-represented. Binding enthalpy is found to be primarily electrostatic, with positively charged amino acids contributing cooperatively to binding enthalpy. Arginine dominates all modes of interaction at the interface. These results suggest that proposed prebiotic syntheses must be compatible with cationic amino acids, particularly arginine or a biophysically similar amino acid, in order to be relevant to the invention of protein by the RNA World.

The function of fitness (or molecular activity) in the space of all possible sequences is known as the fitness landscape. Evolution is a random walk on the fitness landscape, with a bias toward climbing hills. Mapping the topography of real fitness landscapes is fundamental to understanding evolution, but previous efforts were hampered by the difficulty of obtaining large, quantitative data sets. The accessibility of high-throughput sequencing (HTS) has transformed this study, enabling large-scale enumeration of fitness for many mutants and even complete sequence spaces in some cases. We review the progress of high-throughput studies in mapping molecular fitness landscapes, both in vitro and in vivo, as well as opportunities for future research. Such studies are rapidly growing in number. HTS is expected to have a profound effect on the understanding of real molecular fitness landscapes.

The Soai reaction and the Viedma deracemization of racemic conglomerate crystal mixtures are experimental pieces of evidence of the ability of enantioselective autocatalytic coupled networks to yield absolute asymmetric synthesis. Thermodynamically open systems or systems with non-uniform energy distributions may lead to chiral final states and, in systems able to come into thermodynamic equilibrium with their surroundings, to kinetically controlled absolute asymmetric synthesis. The understanding of network parameters and of the thermodynamic scenarios that may lead to spontaneous mirror symmetry breaking (SMSB) could assist in the development of new methods for asymmetric synthesis and enantioselective polymerizations (e.g., replicators), and to frame reasonable speculations on the origin of biological homochirality.

We solve numerically a kinetic model of chiral polymerization in systems closed to matter and energy flow, paying special emphasis to its ability to amplify the small initial enantiomeric excesses due to the internal and unavoidable statistical fluctuations. The reaction steps are assumed to be reversible, implying a thermodynamic constraint among some of the rate constants. Absolute asymmetric synthesis is achieved in this scheme. The system can persist for long times in quasistationary chiral asymmetric states before racemizing. Strong inhibition leads to long-period chiral oscillations in the enantiomeric excesses of the longest homopolymer chains. We also calculate the entropy production σ per unit volume and show that σ increases to a peak value either before or in the vicinity of the chiral symmetry breaking transition.

The model of limited enantioselectivity (LES) in closed systems, and under experimental conditions able to achieve chemical equilibrium, can give rise to neither spontaneous mirror symmetry breaking (SMSB) nor kinetic chiral amplifications. However, it has been recently shown that it is able to lead to SMSB, as a stationary final state, in thermodynamic scenarios involving nonuniform temperature distributions and for compartmentalized separation between the two autocatalytic reactions. Herein, it is demonstrated how SMSB may occur in LES in a cyclic network with uniform temperature distribution if the reverse reaction of the nonenantioselective autocatalysis, which gives limited inhibition on the racemic mixture, is driven by an external reagent, that is, in conditions that keep the system out of chemical equilibrium. The exact stability analysis of the racemic and chiral final outcomes and the study of the reaction parameters leading to SMSB are resolved analytically. Numerical simulations, using chemical kinetics equations, show that SMSB may occur for chemically reasonable parameters. Numerical simulations on SMSB are also presented for speculative, but reasonable, scenarios implying reactions common in amino acid chemistry.