Loading of Amino Acids onto RNA in a Putative RNA-Peptide World

RNA is a molecule that can both store genetic information and perform catalytic reactions. This observed dualism places RNA into the limelight of concepts about the origin of life. The RNA world concept argues that life started from self-replicating RNA molecules, which evolved toward increasingly complex structures. Recently, we demonstrated that RNA, with the help of conserved non-canonical nucleosides, which are also putative relics of an early RNA world, had the ability to grow peptides covalently connected to RNA nucleobases, creating RNA-peptide chimeras. It is conceivable that such molecules, which combined the information-coding properties of RNA with the catalytic potential of amino acid side chains, were once the structures from which life emerged. Herein, we report prebiotic chemistry that enabled the loading of both nucleosides and RNAs with amino acids as the first step toward RNA-based peptide synthesis in a putative RNA-peptide world.     

Can coacervation unify disparate hypotheses in the origin of cellular life?

Here, we review the recent progress in the characterisation and utilisation of coacervates as protocell models in the origin of life studies. We provide evidence that coacervation could have played a unique role during the origin of life, based on its ability to form from a range of different prebiotically relevant molecules; partition solutes; support and alter RNA catalysis and readily deform its shape. We discuss how these properties could have been important for the formation of the first membrane-bound cells, supporting RNA-peptide evolution and primitive metabolism, and in replicating and proliferating by growth and division processes.     

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.     

Ribonucleotides and RNA Promote Peptide Chain Growth

All known forms of life use RNA‐mediated polypeptide synthesis to produce the proteins encoded in their genes. Because the principal parts of the translational machinery consist of RNA, it is likely that peptide synthesis was achieved early in the prebiotic evolution of an RNA‐dominated molecular world. How RNA attracted amino acids and then induced peptide formation in the absence of enzymes has been unclear. Herein, we show that covalent capture of an amino acid as a phosphoramidate favors peptide formation. Peptide coupling is a robust process that occurs with different condensation agents. Kinetics show that covalent capture can accelerate chain growth over oligomerization of the free amino acid by at least one order of magnitude, so that there is no need for enzymatic catalysis for peptide synthesis to begin. Peptide chain growth was also observed on phosphate‐terminated RNA strands. Peptide coupling promoted by ribonucleotides or ribonucleotide residues may have been an important transitional form of peptide synthesis that set in when amino acids were first captured by RNA.     

RNA–DNA Chimeras in the Context of an RNA World Transition to an RNA/DNA World

The RNA world hypothesis posits that DNA and proteins were later inventions of early life, or the chemistry that gave rise to life. Most scenarios put forth for the emergence of DNA assume a clean separation of RNA and DNA polymer, and a smooth transition between RNA and DNA. However, based on the reality of “clutter” and lack of sophisticated separation/discrimination mechanisms in a protobiological (and/or prebiological) world, heterogeneous RNA–DNA backbone containing chimeric sequences could have been common—and have not been fully considered in models transitioning from an RNA world to an RNA–DNA world. Herein we show that there is a significant decrease in Watson–Crick duplex stability of the heterogeneous backbone chimeric duplexes that would impede base‐pair mediated interactions (and functions). These results point to the difficulties for the transition from one homogeneous system (RNA) to another (RNA/DNA) in an RNA world with a heterogeneous mixture of ribo‐ and deoxyribonucleotides and sequences, while suggesting an alternative scenario of prebiological accumulation and co‐evolution of homogeneous systems (RNA and DNA).     

Non-Watson-Crick base pairs in RNA-protein recognition

The cellular functions of most RNA molecules involve protein binding, and non-Watson-Crick base pairs are hallmark sites for interactions with proteins. The determination of three-dimensional structures of RNA-peptide and RNA-protein complexes reveals the molecular basis of non-Watson-Crick base-pair recognition.     

Selection of RNA amide synthases

Background: It is generally accepted that, during evolution, replicating RNA molecules emerged from pools of random polynucleotides. This prebiotic RNA world was followed by an era of RNA-mediated catalysis of amide-bond formation. RNA would thus have provided the machinery responsible for the assembly of peptides and the beginning of the protein world of today. Naturally occurring ribozymes, which catalyze the cleavage or ligation of oligonucleotide phosphodiester bonds, support the idea that RNA could self-replicate. But was RNA constrained to this path and were RNA-acylated carriers required before RNA could catalyze the formation of amide bonds?Results: We have isolated RNA catalysts that are capable of mediating amide-bond synthesis without the need for specifically designed templates to align the substrates, and we have kinetically characterized these catalysts. The rate enhancement observed for these RNA amide synthases exceeds the noncatalyzed amidation rate by a factor of ∼10⁴. In addition, Cu²⁺ ions caused a change in the affinity of RNA for the substrate rather than being directly involved in amide-bond formation.Conclusions: The discovery of these new amide synthases shows how functionally modified nucleic acids can facilitate covalent-bond formation without templating. Previously unforeseen RNA-evolution pathways can, therefore, be considered; for example, to guide amide-bond formation, en route to the protein world, it appears that substrate-binding pockets were formed that are analogous to those of protein enzymes.     

Proto‐Urea‐RNA (Wöhler RNA) Containing Unusually Stable Urea Nucleosides

The RNA world hypothesis assumes that life on Earth began with nucleotides that formed information‐carrying RNA oligomers able to self‐replicate. Prebiotic reactions leading to the contemporary nucleosides are now known, but their execution often requires specific starting materials and lengthy reaction sequences. It was therefore proposed that the RNA world was likely proceeded by a proto‐RNA world constructed from molecules that were likely present on the early Earth in greater abundance. Herein, we show that the prebiotic starting molecules bis‐urea (biuret) and tris‐urea (triuret) are able to directly react with ribose. The urea‐ribosides are remarkably stable because they are held together by a network of intramolecular, bifurcated hydrogen bonds. This even allowed the synthesis of phosphoramidite building blocks and incorporation of the units into RNA. Investigations of the nucleotides’ base‐pairing potential showed that triuret:G RNA base pairs closely resemble U:G wobble base pairs. Based on the probable abundance of urea on the early Earth, we postulate that urea‐containing RNA bases are good candidates for a proto‐RNA world.     

In vitro selection of highly modified cyclic peptides that act as tight binding inhibitors

There is a great demand for the discovery of new therapeutic molecules that combine the high specificity and affinity of biologic drugs with the bioavailability and lower cost of small molecules. Small, natural-product-like peptides hold great promise in bridging this gap; however, access to libraries of these compounds has been a limitation. Since ribosomal peptides may be subjected to in vitro selection techniques, the generation of extremely large libraries (>10(13)) of highly modified macrocyclic peptides may provide a powerful alternative for the generation and selection of new useful bioactive molecules. Moreover, the incorporation of many non-proteinogenic amino acids into ribosomal peptides in conjunction with macrocyclization should enhance the drug-like features of these libraries. Here we show that mRNA-display, a technique that allows the in vitro selection of peptides, can be applied to the evolution of macrocyclic peptides that contain a majority of unnatural amino acids. We describe the isolation and characterization of two such unnatural cyclic peptides that bind the protease thrombin with low nanomolar affinity, and we show that the unnatural residues in these peptides are essential for the observed high-affinity binding. We demonstrate that the selected peptides are tight-binding inhibitors of thrombin, with K(i)(app) values in the low nanomolar range. The ability to evolve highly modified macrocyclic peptides in the laboratory is the first crucial step toward the facile generation of useful molecular reagents and therapeutic lead molecules that combine the advantageous features of biologics with those of small-molecule drugs.     

Stepwise molding of a highly selective ribonucleopeptide receptor

The structural characteristics of RNA-peptide (RNP) complexes are suitable for molding of a ligand-binding pocket of the RNP complex in a stepwise manner. The first step involves molding of the RNA subunit by in vitro selection of an RNP pool originating from an RNA library and the peptide, as previously reported for the construction of an ATP-binding RNP complex from an RRE RNA-Rev peptide complex. The second step involves selection from an RNP library consisting of Rev peptides with randomized amino acid residues and the RNA subunit selected in the first molding. The ATP-binding pocket produced by sequential molding of RNA and peptide subunits shows higher affinity to ATP and a distinct specificity for ATP versus dATP as compared to the ATP-binding RNP receptor in which only the RNA subunit has been molded. The second step selection from the peptide-based RNP library allows expansion of the ATP recognition surface, consisting of both RNA and peptide subunits, to enhance the affinity and selectivity to discriminate ATP against dATP. Our approach of stepwise molding offers the advantage of increasing the diversity of the RNP library by utilizing characteristics of different biopolymers. The ribonucleopeptide-based, multi-subunit approach is also extendable to other biomacromolecular assemblies, which may yield artificial receptors and enzymes with increased specificity and more diverse chemical activities.     

Noncanonical hydrogen bonding in nucleic acids. Benchmark evaluation of key base-phosphate interactions in folded RNA molecules using quantum-chemical calculations and molecular dynamics simulations

RNA molecules are stabilized by a wide range of noncanonical interactions that are not present in DNA. Among them, the recently classified base-phosphate (BPh) interactions belong to the most important ones. Twelve percent of nucleotides in the ribosomal crystal structures are involved in BPh interactions. BPh interactions are highly conserved and provide major constraints on RNA sequence evolution. Here we provide assessment of the energetics of BPh interactions using MP2 computations extrapolated to the complete basis set of atomic orbitals and corrected for higher-order electron correlation effects. The reference computations are compared with DFT-D and DFT-D3 approaches, the SAPT method, and the molecular mechanics force field. The computations, besides providing the basic benchmark for the BPh interactions, allow some refinements of the original classification, including identification of some potential doubly bonded BPh patterns. The reference computations are followed by analysis of some larger RNA fragments that consider the context of the BPh interactions. The computations demonstrate the complexity of interaction patterns utilizing the BPh interactions in real RNA structures. The BPh interactions are often involved in intricate interaction networks. We studied BPh interactions of protonated adenine that can contribute to catalysis of hairpin ribozyme, the key BPh interaction in the S-turn motif of the sarcin-ricin loop, which may predetermine the S-turn topology and complex BPh patterns from the glmS riboswitch. Finally, the structural stability of BPh interactions in explicit solvent molecular dynamics simulations is assessed. The simulations well preserve key BPh interactions and allow dissection of structurally/functionally important water-meditated BPh bridges, which could not be considered in earlier bioinformatics classification of BPh interactions.     

Possible steps to the emergence of life: the [GADV]-protein world hypothesis

Based on the fact that RNA has not only a genetic function but also a catalytic function, the RNA world theory on the origin of life was first proposed about 20 years ago. The theory assumes that RNA was amplified by self-replication to increase RNA diversity on the primitive earth. Since then, the theory has been widely accepted as the most likely explanation for the emergence of life. In contrast, we reached another hypothesis, the [GADV]-protein world hypothesis, which is based on pseudo-replication of [GADV]-proteins. We reached this hypothesis during studies on the origins of genes and the genetic code, where [G], [A], [D], and [V] refer to Gly, Ala, Asp, and Val, respectively. In this review, possible steps to the emergence of life are discussed from the standpoint of the [GADV]-protein world hypothesis, comparing it in parallel with the RNA world theory. It is also shown that [GADV]-peptides, which were produced by repeated dry-heating cycles and by solid phase peptide synthesis, have catalytic activities, hydrolyzing peptide bonds in a natural protein, bovine serum albumin. These experimental results support the [GADV]-protein world hypothesis for the origin of life.     

On the Evolutionary History of the Twenty Encoded Amino Acids

α-Amino acids are essential molecular constituents of life, twenty of which are privileged because they are encoded by the ribosomal machinery. The question remains open as to why this number and why this 20 in particular, an almost philosophical question that cannot be conclusively resolved. They are closely related to the evolution of the genetic code and whether nucleic acids, amino acids, and peptides appeared simultaneously and were available under prebiotic conditions when the first self-sufficient complex molecular system emerged on Earth. This report focuses on prebiotic and metabolic aspects of amino acids and proteins starting with meteorites, followed by their formation, including peptides, under plausible prebiotic conditions, and the major biosynthetic pathways in the various kingdoms of life. Coenzymes play a key role in the present analysis in that amino acid metabolism is linked to glycolysis and different variants of the tricarboxylic acid cycle (TCA, rTCA, and the incomplete horseshoe version) as well as the biosynthesis of the most important coenzymes. Thus, the report opens additional perspectives and facets on the molecular evolution of primary metabolism.     

Spontaneous Formation of RNA Strands,Peptidyl RNA,and Cofactors

How the biochemical machinery evolved from simple precursors is an open question. Here we show that ribonucleotides and amino acids condense to peptidyl RNAs in the absence of enzymes under conditions established for genetic copying. Untemplated formation of RNA strands that can encode genetic information, formation of peptidyl chains linked to RNA, and formation of the cofactors NAD⁺, FAD, and ATP all occur under the same conditions. In the peptidyl RNAs, the peptide chains are phosphoramidate‐linked to a ribonucleotide. Peptidyl RNAs with long peptide chains were selected from an initial pool when a lipophilic phase simulating the interior of membranes was offered, and free peptides were released upon acidification. Our results show that key molecules of genetics, catalysis, and metabolism can emerge under the same conditions, without a mineral surface, without an enzyme, and without the need for chemical pre‐activation.     

从生活世界到科学世界——新课程理念下化学教学范式的重要转型

把化学带到生活中去,加强与社会生活的联系,使化学教学贴近学生的社会生活实际,让学生以社会生活为背景学习化学知识,并使其了解化学在现代社会生活中的价值,是20世纪90年代以来世界中学化学课程改革的重要趋势,也是当前我国基础教育课程改革中化学教学范式的重要转型。落实从生活世界向科学世界的化学教学范式的转变,完整地体现化学课程的价值,是当前我国中学化学课程改革所面临的一项重要任务。     

Minimum sequence requirements for selective RNA-ligand binding: a molecular mechanics algorithm using molecular dynamics and free-energy techniques

In vitro evolution techniques allow RNA molecules with unique functions to be developed. However, these techniques do not necessarily identify the simplest RNA structures for performing their functions. Determining the simplest RNA that binds to a particular ligand is currently limited to experimental protocols. Here, we introduce a molecular-mechanics based algorithm employing molecular dynamics simulations and free-energy methods to predict the minimum sequence requirements for selective ligand binding to RNA. The algorithm involves iteratively deleting nucleotides from an experimentally determined structure of an RNA-ligand complex, performing energy minimizations and molecular dynamics on each truncated structure, and assessing which truncations do not prohibit RNA binding to the ligand. The algorithm allows prediction of the effects of sequence modifications on RNA structural stability and ligand-binding energy. We have implemented the algorithm in the AMBER suite of programs, but it could be implemented in any molecular mechanics force field parameterized for nucleic acids. Test cases are presented to show the utility and accuracy of the methodology.     

Chemistry and the missing era of evolution

The Darwinian evolution of life on earth depends utterly on complex molecular machinery, which, it seems, could only have arisen through a Darwinian evolution. The "RNA world" idea reduces this paradox, but requires a geochemically implausible supply of RNA monomers. A pre-RNA era of natural selection is implied. I suggest that originally this was based on inorganic materials that came to replicate permutations with specific (e.g. catalytic) effects.     

Noncanonical RNA Nucleosides as Molecular Fossils of an Early Earth—Generation by Prebiotic Methylations and Carbamoylations

The RNA‐world hypothesis assumes that life on Earth started with small RNA molecules that catalyzed their own formation. Vital to this hypothesis is the need for prebiotic routes towards RNA. Contemporary RNA, however, is not only constructed from the four canonical nucleobases (A, C, G, and U), it also contains many chemically modified (noncanonical) bases. A still open question is whether these noncanonical bases were formed in parallel to the canonical bases (chemical origin) or later, when life demanded higher functional diversity (biological origin). Here we show that isocyanates in combination with sodium nitrite establish methylating and carbamoylating reactivity compatible with early Earth conditions. These reactions lead to the formation of methylated and amino acid modified nucleosides that are still extant. Our data provide a plausible scenario for the chemical origin of certain noncanonical bases, which suggests that they are fossils of an early Earth.     

Click Chemistry Route to the Synthesis of Unusual Amino Acids,Peptides, Triazole‐Fused Heterocycles and Pseudodisaccharides

Conjugation of different molecular species using copper(I)‐catalyzed click reaction between azides and terminal alkynes is among the best available methods to prepare multifunctional compounds. The effectiveness of this method has provided wider acceptance to the concept of click chemistry, which is now widely employed to synthesize densely functionalized organic molecules. This article summarizes the contributions from our group in the development of new methods for the synthesis of functional molecules using copper(I)‐catalyzed click reactions. We have developed very efficient methods for the synthesis of peptides and amino acids conjugated with carbohydrates, thymidine and ferrocene. We have also developed an efficient strategy to synthesize triazole‐fused heterocycles from primary amines, amino alochols and diols. Finally, an interesting method for the synthesis of pseudodisaccharides linked through triazoles, starting from carbohydrate‐derived donor‐acceptor cyclopropanes is discussed.     

A bridge between the RNA and protein worlds? Accelerating delivery of chemical reactivity to RNA and DNA by a specific short peptide (AAKK)(4)

BACKGROUND: RNA can catalyze diverse chemical reactions, leading to the hypothesis that an RNA world existed early in evolution. Today, however, catalysis by naturally occurring RNAs is rare and most chemical transformations within cells require proteins. This has led to interest in the design of small peptides capable of catalyzing chemical transformations. RESULTS: We demonstrate that a short lysine-rich peptide (AAKK)(4) can deliver a nucleophile to DNA or RNA and amplify the rate of chemical modification by up to 3400-fold. We also tested similar peptides that contain ornithine or arginine in place of lysine, peptides with altered stereochemistry or orientation, and peptides containing eight lysines but with different spacing. Surprisingly, these similar peptides function much less well, suggesting that specific combinations of amino acids, charge distribution, and stereochemistry are necessary for the rate enhancement by (AAKK)(4). CONCLUSIONS: By appending other reactive groups to (AAKK)(4) it should be possible to greatly expand the potential for small peptides to directly catalyze modification of DNA or RNA or to act as cofactors to promote ribozyme catalysis.