Forty years of in vitro evolution

It has been 40 years since Spiegelman and co-workers demonstrated how RNA molecules can be evolved in the test tube. This result established Darwinian evolution as a chemical process and paved the way for the many directed evolution experiments that followed. Chemists can benefit from reflecting on Spiegelman's studies and the subsequent advances, which have taken the field to the brink of the generation of life itself in the laboratory. This Review summarizes the concepts and methods for the directed evolution of RNA molecules in vitro.     

Darwinian Docking

The Darwinian model of evolution is an optimization strategy that can be adapted to docking. It differs from the common use of genetic algorithms, primarily in its acceptance of diverse solutions over finding "global" optima. A related problem is selecting compounds using multiple criteria. I discuss these ideas and present the outlines of a protocol for selecting "hits" and "leads" in drug discovery.     

Amino Acid Modified RNA Bases as Building Blocks of an Early Earth RNA-Peptide World

Fossils of extinct species allow us to reconstruct the process of Darwinian evolution that led to the species diversity we see on Earth today. The origin of the first functional molecules able to undergo molecular evolution and thus eventually able to create life, are largely unknown. The most prominent idea in the field posits that biology was preceded by an era of molecular evolution, in which RNA molecules encoded information and catalysed their own replication. This RNA world concept stands against other hypotheses, that argue for example that life may have begun with catalytic peptides and primitive metabolic cycles. The question whether RNA or peptides were first is addressed by the RNA-peptide world concept, which postulates a parallel existence of both molecular species. A plausible experimental model of how such an RNA-peptide world may have looked like, however, is absent. Here we report the synthesis and physicochemical evaluation of amino acid containing adenosine bases, which are closely related to molecules that are found today in the anticodon stem-loop of tRNAs from all three kingdoms of life. We show that these adenosines lose their base pairing properties, which allow them to equip RNA with amino acids independent of the sequence context. As such we may consider them to be living molecular fossils of an extinct molecular RNA-peptide world.     

Continuous in vitro evolution of a ribozyme that catalyzes three successive nucleotidyl addition reactions

Variants of the class I ligase ribozyme, which catalyzes joining of the 3' end of a template bound oligonucleotide to its own 5' end, have been made to evolve in a continuous manner by a simple serial transfer procedure that can be carried out indefinitely. This process was expanded to allow the evolution of ribozymes that catalyze three successive nucleotidyl addition reactions, two template-directed mononucleotide additions followed by RNA ligation. During the development of this behavior, a population of ribozymes was maintained against an overall dilution of more than 10(406). The resulting ribozymes were capable of catalyzing the three-step reaction pathway, with nucleotide addition occurring in either a 5'-->3' or a 3'-->5' direction. This purely chemical system provides a functional model of a multi-step reaction pathway that is undergoing Darwinian evolution.     

A genetic algorithm for the automated generation of small organic molecules: Drug design using an evolutionary algorithm

Rational drug design involves finding solutions to large combinatorial problems for which an exhaustive search is impractical. Genetic algorithms provide a novel tool for the investigation of such problems. These are a class of algorithms that mimic some of the major characteristics of Darwinian evolution. LEA has been designed in order to conceive novel small organic molecules which satisfy quantitative structure-activity relationship based rules (fitness). The fitness consists of a sum of constraints that are range properties. The algorithm takes an initial set of fragments and iteratively improves them by means of crossover and mutation operators that are related to those involved in Darwinian evolution. The basis of the algorithm, its implementation and parameterization, are described together with an application in de novo molecular design of new retinoids. The results may be promising for chemical synthesis and show that this tool may find extensive applications in de novo drug design projects.     

Virtual darwinian drug design: QSAR inverse problem, virtual combinatorial chemistry, and computational screening

The generation of diversity and its further selection by an external system is a common mechanism for the evolution of the living species and for the current drug design methods. This assumption allows us to label the methods based on generation and selection of molecular diversity as "Darwinian" ones, and to distinguish them from the structure-based, structure-modulation approaches. An example of a Darwinian method is the inverse QSAR. It consists of the computational generation of candidate chemical structures and their selection according to a previously established QSAR model. New trends in the field of combinatorial chemical syntheses comprise the concepts of virtual combinatorial synthesis and virtual or computational screening. Virtual combinatorial synthesis, closely related to inverse QSAR, can be defined as the computational simulation of the generation of new chemical structures by using a combinatorial strategy to generate a virtual library. Virtual screening is the selection of chemical structures having potential desirable properties from a database or virtual library in order to be synthesized and assayed. This review is mainly focused on graph theoretical drug design approaches, but a survey with key references is provided that covers other simulation methods.     

Theory, modelling and simulation in origins of life studies

Origins of life studies represent an exciting and highly multidisciplinary research field. In this review we focus on the contributions made by theory, modelling and simulation to addressing fundamental issues in the domain and the advances these approaches have helped to make in the field. Theoretical approaches will continue to make a major impact at the "systems chemistry" level based on the analysis of the remarkable properties of nonlinear catalytic chemical reaction networks, which arise due to the auto-catalytic and cross-catalytic nature of so many of the putative processes associated with self-replication and self-reproduction. In this way, we describe inter alia nonlinear kinetic models of RNA replication within a primordial Darwinian soup, the origins of homochirality and homochiral polymerization. We then discuss state-of-the-art computationally-based molecular modelling techniques that are currently being deployed to investigate various scenarios relevant to the origins of life.     

Does Pressure Break Mirror-Image Symmetry? A Perspective and New Insights

This paper is aimed at dissecting and discussing the effect of high pressure on chirogenesis, thus unveiling the role of this universal force in astrochemical and primeval Darwinian scenarios. The first part of this contribution revisits the current status and recent experiments, most dealing with crystalline racemates, for which generation of metastable conglomeratic phases would eventually afford spontaneous resolution and hence enantioenriched mixtures. We then provide an in-depth thermodynamic analysis, based on previous studies of non-electrolyte solutions and dense mixtures accounting for the existence of positive excess volume upon mixing, to simulate the mirror symmetry breaking, the evolution of entropy production and dissipation due to enantiomer conversion. Results clearly suggest that mirror symmetry breaking under high pressure may be a genuine phenomenon and that enantioenrichment from initial scalemic mixtures may also take place.     

A ribozyme for the aldol reaction

Directed in vitro evolution can create RNA catalysts for a variety of organic reactions, supporting the "RNA world" hypothesis, which proposes that metabolic transformations in early life were catalyzed by RNA molecules rather than proteins. Among the most fundamental carbon-carbon bond-forming reactions in nature is the aldol reaction, mainly catalyzed by aldolases that utilize either an enamine mechanism (class I) or a Zn(2+) cofactor (class II). We report on isolation of a Zn(2+)-dependent ribozyme that catalyzes an aldol reaction at its own modified 5' end with a 4300-fold rate enhancement over the uncatalyzed background reaction. The ribozyme can also act as an intermolecular catalyst that transfers a biotinylated benzaldehyde derivative to the aldol donor substrate, coupled to an external hexameric RNA oligonucleotide, supporting the existence of RNA-originated biosynthetic pathways for metabolic sugar precursors and other biomolecules.     

Evaporite Borate‐Containing Mineral Ensembles Make Phosphate Available and Regiospecifically Phosphorylate Ribonucleosides: Borate as a Multifaceted Problem Solver in Prebiotic Chemistry

RNA is currently thought to have been the first biopolymer to support Darwinian natural selection on Earth. However, the phosphate esters in RNA and its precursors, and the many sites at which phosphorylation might occur in ribonucleosides under conditions that make it possible, challenge prebiotic chemists. Moreover, free inorganic phosphate may have been scarce on early Earth owing to its sequestration by calcium in the unreactive mineral hydroxyapatite. Herein, it is shown that these problems can be mitigated by a particular geological environment that contains borate, magnesium, sulfate, calcium, and phosphate in evaporite deposits. Actual geological environments, reproduced here, show that Mg²⁺ and borate sequester phosphate from calcium to form the mineral lüneburgite. Ribonucleosides stabilized by borate mobilize borate and phosphate from lüneburgite, and are then regiospecifically phosphorylated by the mineral. Thus, in addition to guiding carbohydrate pre‐metabolism, borate minerals in evaporite geoorganic contexts offer a solution to the phosphate problem in the “RNA first” model for the origins of life.     

DNA-Encoded Libraries: Towards Harnessing their Full Power with Darwinian Evolution

DNA-encoded library (DEL) technologies are transforming the drug discovery process, enabling the identification of ligands at unprecedented speed and scale. DEL makes use of libraries that are orders of magnitude larger than traditional high-throughput screens. While a DNA tag alludes to a genotype–phenotype connection that is exploitable for molecular evolution, most of the work in the field is performed with libraries where the tag serves as an amplifiable barcode but does not allow “translation” into the synthetic product it is linked to. In this Review, we cover technologies that enable the “translation” of the genetic tag into synthetic molecules, both biochemically and chemically, and explore how it can be used to harness Darwinian evolutionary pressure.     

Out of fuzzy chemistry: from prebiotic chemistry to metabolic networks

The origin of life on Earth was a chemical affair. So how did primitive biochemical systems originate from geochemical and cosmochemical processes on the young planet? Contemporary research into the origins of life subscribes to the Darwinian principle of material causes operating in an evolutionary context, as advocated by A. I. Oparin and J. B. S. Haldane in the 1920s. In its simplest form (e.g., a bacterial cell) extant biological complexity relies on the functional integration of metabolic networks and replicative genomes inside a lipid boundary. Different research programmes have explored the prebiotic plausibility of each of these autocatalytic subsystems and combinations thereof: self-maintained networks of small molecules, template chemistry, and self-reproductive vesicles. This tutorial review focuses on the debates surrounding the origin of metabolism and offers a brief overview of current studies on the evolution of metabolic networks. I suggest that a leitmotif in the origin and evolution of metabolism is the role played by catalysers' substrate ambiguity and multifunctionality.     

Investigating and Engineering Enzymes by Genetic Selection

Natural enzymes have arisen over millions of years by the gradual process of Darwinian evolution. The fundamental steps of evolution-mutation, selection, and amplification-can also be exploited in the laboratory to create and characterize protein catalysts on a human timescale. In vivo genetic selection strategies enable the exhaustive analysis of protein libraries with 10(10) different members, and even larger ensembles can be studied with in vitro methods. Evolutionary approaches can consequently yield statistically meaningful insight into the complex and often subtle interactions that influence protein folding, structure, and catalytic mechanism. Such methods are also being used increasingly as an adjunct to design, thus providing access to novel proteins with tailored catalytic activities and selectivities.     

Quantitative analysis of receptors for adenosine nucleotides obtained via in vitro selection from a library incorporating a cationic nucleotide analog.

5-(3"-Aminopropynyl)-2'-deoxyuridine (dJ), a modified nucleoside with a side chain carrying a cationic functional group, was incorporated into an oligonucleotide library, which was amplified using the Vent DNA polymerase in a polymerase chain reaction (PCR). When coupled to an in vitro selection procedure, PCR amplification generated receptors that bind ATP. This is the first example of an in vitro selection generating oligonucleotide receptors where the oligonucleotide library has incorporated a cationic nucleotide functionality. The selection yielded functionalized receptors having sequences differing from a motif known to arise in a standard selection experiment using only natural nucleotides. Surprisingly, both the natural and the functionalized motifs convergently evolved to bind not one, but two ATP molecules cooperatively. Likewise, the affinity of the receptors for ATP had converged; in both cases, the receptors are half saturated at the 3 mM concentrations of ATP presented during the selection. The convergence of phenotype suggests that the outcome of this selection experiment was determined by features of the environment during which selection occurs, in particular, a highly loaded affinity resin used in the selection step. Further, the convergence of phenotype suggests that the optimal molecular phenotype has been achieved by both selections for the selection conditions. This interplay between environmental conditions demanding a function of a biopolymer and the ability of the biopolymer to deliver that function is strictly analogous to that observed during natural selection, illustrating the nature of life as a self-sustaining chemical system capable of Darwinian evolution.     

RNA sex

Recombination of genetic information is a major driving force in evolution, today catalyzed by protein enzymes. In this issue of Chemistry & Biology, a paper by Riley and Lehman demonstrates that RNA can perform general recombination of RNA strands, thus supporting the scenario of a prebiotic RNA world.     

Electrostatic Localization of RNA to Protocell Membranes by Cationic Hydrophobic Peptides

Cooperative interactions between RNA and vesicle membranes on the prebiotic earth may have led to the emergence of primitive cells. The membrane surface offers a potential platform for the catalysis of reactions involving RNA, but this scenario relies upon the existence of a simple mechanism by which RNA could become associated with protocell membranes. Here, we show that electrostatic interactions provided by short, basic, amphipathic peptides can be harnessed to drive RNA binding to both zwitterionic phospholipid and anionic fatty acid membranes. We show that the association of cationic molecules with phospholipid vesicles can enhance the local positive charge on a membrane and attract RNA polynucleotides. This phenomenon can be reproduced with amphipathic peptides as short as three amino acids. Finally, we show that peptides can cross bilayer membranes to localize encapsulated RNA. This mechanism of polynucleotide confinement could have been important for primitive cellular evolution.     

Genetics first or metabolism first? The formamide clue

Life is made of the intimate interaction of metabolism and genetics, both built around the chemistry of the most common elements of the Universe (hydrogen, oxygen, nitrogen, and carbon). The transmissible interaction of metabolic and genetic cycles results in the hypercycles of organization and de-organization of chemical information, of living and non-living. The origin-of-life quest has long been split into several attitudes exemplified by the aphorisms "genetics-first" or "metabolism-first". Recently, the opposition between these approaches has been solved by more unitary theoretical and experimental frames taking into account energetic, evolutionary, proto-metabolic and environmental aspects. Nevertheless, a unitary and simple chemical frame is still needed that could afford both the precursors of the synthetic pathways eventually leading to RNA and to the key components of the central metabolic cycles, possibly connected with the synthesis of fatty acids. In order to approach the problem of the origin of life it is therefore reasonable to start from the assumption that both metabolism and genetics had a common origin, shared a common chemical frame, and were embedded under physical-chemical conditions favourable for the onset of both. The singleness of such a prebiotically productive chemical process would partake of Darwinian advantages over more complex fragmentary chemical systems. The prebiotic chemistry of formamide affords in a single and simple physical-chemical frame nucleic bases, acyclonucleosides, nucleotides, biogenic carboxylic acids, sugars, amino sugars, amino acids and condensing agents. Thus, we suggest the possibility that formamide could have jointly provided the main components for the onset of both (pre)genetic and (pre)metabolic processes. As a note of caution, we discuss the fact that these observations only indicate possible solutions at the level of organic substrates, not at the systemic chemical level.     

An efficient thermally induced RNA conformational switch as a framework for the functionalization of RNA nanostructures

RNA offers a variety of interactions and dynamic conformational switches not available with DNA that may be exploited for the construction of nanomolecular structures. Here, we show how the RNA loop-loop, or "kissing", interaction can be used to construct specific circular RNA arrangements that are capable of thermal isomerization to alternative structures. We also show how this thermally induced structural rearrangement can be used to unmask a functional RNA structure, in this case, a peptide-binding RNA structure, the Rev-response element (RRE) of HIV, thereby acting as a functional peptide-binding switch. The relative ease with which the RRE could be engineered into the RNA substrates suggested that a variety of functional RNA structures may be introduced. In addition, the structural rearrangement was extremely efficient, showing that the "kissing" complexes described in this study may provide a useful framework for the construction of functional RNA-based nanostructures, as well as aid in our understanding of the way RNA functions in biological systems.     

Non-Enzymatic RNA Backbone Proofreading through Energy-Dissipative Recycling

Non-enzymatic oligomerization of activated ribonucleotides leads to ribonucleic acids that contain a mixture of 2′,5′- and 3′,5′-linkages, and overcoming this backbone heterogeneity has long been considered a major limitation to the prebiotic emergence of RNA. Herein, we demonstrate non-enzymatic chemistry that progressively converts 2′,5′-linkages into 3′,5′-linkages through iterative degradation and repair. The energetic costs of this proofreading are met by the hydrolytic turnover of a phosphate activating agent and an acylating agent. With multiple rounds of this energy-dissipative recycling, we show that all-3′,5′-linked duplex RNA can emerge from a backbone heterogeneous mixture, thereby delineating a route that could have driven RNA evolution on the early earth.