Selectivity of montmorillonite catalyzed prebiotic reactions of D,L-nucleotides

The montmorillonite-catalyzed reactions of the 5′-phosphorimidazolides of D, L-adenosine (D, L-ImpA) (Figure 1a. N = A, R = H) and D, L-uridine (Figure 1a., N = U, R = H) yields oligomers that were as long as 7 mers and 6 mers, respectively. The reactions of dilute solutions of D-ImpA and D-ImpU under the same conditions gave oligomers as long as 9 and 8 mers respectively. This demonstrated that oligomer formation is only partially inhibited by incorporation of both the D- and L-enantiomers. The structures of the dimers, trimers and tetramer fractions formed from D, L-ImpA was investigated by selective enzymatic hydrolysis, comparison with authentic samples and mass spectrometry. Homochiral products were present in greater amounts than would be expected if theoretical amounts of each were formed. The ratio of the proportion of homochiral products to that of the amount of each expected for the dimers (cyclic and linear), trimers and tetramers, was 1.3, 1.6, and 2.1, respectively. In the D, L-ImpU reaction homochiral products did not predominate with ratios of dimers (cyclic and linear), trimers and tetramers 0.8, 0.44, and 1.4, respectively. The proportions of cyclic dimers in the dimer fraction were 52–66% with D, L-ImpA and 44–69% with D, L-ImpU. No cyclic dimers were formed in the absence of montmorillonite. The differences in the reaction products of D, L-ImpA and D, L-ImpU are likely to be due to the difference in the orientations of the activated monomers when bound to the catalytic sites on montmorillonite. The consequences of the selectivity of montmorillonite as a prebiotic catalyst are discussed.     

Effect of Inhibitors on the Montmorillonite Clay-Catalyzed Formation of RNA: Studies on the Reaction Pathway

The Langmuir adsorption isotherms of the phosphoroimidazolides of adenosine (ImpA) and uridine (ImpU), dA^5'ppdA and N⁶, N⁶-dimethyladenine binding on montmorillonite are consistent with their forming a monolayer on the clay surface. This suggests the condensation of ImpA and ImpU to oligomers proceeds on the surface of the clay and not in groups of monomers stacked on the clay surfaces. The binding and reactions of ImpU and ImpA on montmorillonite are blocked by N⁶, N⁶-dimethyladenine and dA^5'ppdA. dA^5'ppdA is a better inhibitor of oligomer formation than N⁶, N⁶-dimethyladenine because both adenine rings of dA^5'ppdA bind to the clay surface and block adjacent catalytic sites. An upper limit of 5–10 × 10¹⁵catalytic sites on 50 mg of clay was estimated from the binding of ImpU and the inhibition of oligomer formation by dA^5'ppdA.     

An Interpretive Review of the Origin of Life Research

Life appears to be a natural property of matter, but the problem of its origin only arose after early scientists refuted continuous spontaneous generation. There is no chance of life arising ‘all at once’, we need the standard scientific incremental explanation with large numbers of small steps, an approach used in both physical and evolutionary sciences. The necessity for considering both theoretical and experimental approaches is emphasized. After describing basic principles that are available (including the Darwin-Eigen cycle), the search for origins is considered under four main themes. These are the RNA-world hypothesis; potential intermediates between an RNA-world and a modern world via the evolution of protein synthesis and then of DNA; possible alternatives to an RNA-world; and finally the earliest stages from the simple prebiotic systems to RNA. The triplicase/proto-ribosome theory for the origin of the ribosome is discussed where triples of nucleotides are added to a replicating RNA, with the origin of a triplet code well-before protein synthesis begins. The length of the code is suggested to arise from the early development of a ratchet mechanism that overcomes the problem of continued processivity of an RNA-based RNA-polymerase. It is probable that there were precursor stages to RNA with simpler sugars, or just two nucleotides, but we do not yet know of any better alternatives to RNA that were likely to arise naturally. For prebiotic stages (before RNA) a flow-reactor model is suggested to solve metabolism, energy gradients, and compartmentation simultaneously – thus the intense interest in some form of flow reactor. If an autocatalytic cycle could arise in such a system we would be major steps ahead. The most likely physical conditions for the origin of life require further clarification and it is still unclear whether the origin of life is more of an entropy (information) problem (and therefore high temperatures would be detrimental), rather than a kinetic problem (where high temperatures may be advantageous).     

Intramolecular RNA replicase: Possibly the first self-replicating molecule in the RNA world

Although there is more and more evidence suggested the existence of an RNA World during the origin of life, the scenario concerning the origin of the RNA World remains blurry. Usually it is speculated that it originated from a prebiotic nucleotide pool, during which a self-replicating RNA synthesis ribozyme may have emerged as the first ribozyme – the RNA replicase. However, there is yet no ersuasive supposition for the mechanism for the self-favouring feature of the replicase, thus the speculation remains unconvincing. Here we suggest that intramolecular catalysis is a possible solution. Two RNA synthesis ribozymes may be integrated into one RNA molecule, as two functional domains which could catalyze the copy of each other. Thus the RNA molecule could self-replicate and be referred to as “intramolecular replicase“ here. Computational simulation to get insight into the dynamic mechanism of emergence of the intramolecular replicase from a nucleotide pool is valuable and would be included in a following work of our group.     

RNA Folding Argues Against a Hot-Start Origin of Life

Opinion is strongly divided on whether life arose on earth under hot or cold conditions, the hot-start and cold-start scenarios, respectively. The origin of life close to deep thermal vents appears as the majority opinion among biologists, but there is considerable biochemical evidence that high temperatures are incompatible with an RNA world. To be functional, RNA has to fold into a three-dimensional structure. We report both theoretical and experimental results on RNA folding and show that (as expected) hot conditions strongly reduce RNA folding. The theoretical results come from energy-minimization calculations of the average extent of folding of RNA, mainly from 0–90°C, for both random sequences and tRNA sequences. The experimental results are from circular-dichroism measurements of tRNA over a similar range of temperatures. The quantitative agreement between calculations and experiment is remarkable, even to the shape of the curves indicating the cooperative nature of RNA folding and unfolding. These results provide additional evidence for a lower temperature stage being necessary in the origin of life. Received: 1 March 2000 / Accepted: 14 June 2000     

RNA Ligation and the Origin of tRNA

A straightforward origin of transfer RNA,(tRNA), is difficult to envision because of the apparentlycomplex idiosyncratic interaction between the D-loop and T-loop. Recently, multiple examples of the T-loop structuralmotif have been identified in ribosomal RNA. These examplesshow that the long-range interactions between the T-loop andD-loops seen in tRNA are not an essential part of the motifbut rather are facilitated by it. Thus, the core T-loopstructure could already have existed in a small RNA prior tothe emergence of the tRNA. The tRNA might then have arisenby expansion of an RNA that carried the motif. With thisidea in mind, Di Giulio's earlier hypothesis that tRNAevolved by a simple duplication or ligation of a minihelixRNA was re-examined. It is shown that an essentially moderntRNA structure can in fact be generated by the ligation oftwo 38-nucleotide RNA minihelices of appropriate sequence.Although rare, such sequences occur with sufficientfrequency, (1 in 3 × 10⁷), that they could be found in astandard in vitro RNA selection experiment. Theresults demonstrate that a series of RNA duplications, aspreviously proposed, can in principal account for the originof tRNA. More generally, the results point out that RNAligation can be a powerful driving force for increasedcomplexity in the RNA World.     

RNA synthesis of vesicular stomatitis virus. VIII. Oligonucleotides of the structural genes and mRNA.

The single-stranded RNA genome of vesicular stomatitis virus (VSV, Indiana serotype, San Juan strain) yields approx. 75 RNase T1-resistant oligonucleotides ranging in size from 10 to 50 bases. Each of the five structural genes, isolated as duplex RNA molecules hybridized to complementary mRNA, contains two or more of these large oligonucleotides. One of the oligonucleotides is identified as part of the non-coding region near the 3' end of the genome. Comparison of these results with others indicate that the RNA sequence of VSV is apparently stable in the laboratory but not in the wild. RNase T1-resistant oligonucleotides are also shown for all five VSV mRN species. Whether the mRNA for these digestions are are isolated from duplex RNA molecules or as single-stranded RNA species, the oligonucleotide patterns for each mRNA are virtually identical, indicating that each mRNA is transcribed from contiguous sequences on the genome. Comparison with published oligonucleotide patterns obtained from other isolates of VSV or from VSV deletion mutants indicate that identity and changes in their genome structure can be correlated with specific structural genes.     

Thermal Energy and the Origin of Life

Life has evolved on Earth with electromagnetic radiation (light), fermentable organic molecules, and oxidizable chemicals as sources of energy. Biological use of thermal energy has not been observed although heat, and the thermal gradients required to convert it into free energy, are ubiquitous and were even more abundant at the time of the origin of life on Earth. Nevertheless, Earth-organisms sense thermal energy, and in suitable environments may have gained the capability to use it as energy source. It has been proposed that the first organisms obtained their energy by a first protein named pF₁ that worked on a thermal variation of the binding change mechanism of today's ATP sythase enzyme. Organisms using thermosynthesis may still live where light or chemical energy sources are not available. Possible suitable examples are subsurface environments on Earth and in the outer Solar System, in particular the subsurface oceans of the icy satellites of Jupiter and Saturn.     

A Research Proposal on the Origin Of Life. Closing Lecture given at the ISSOL Congress in Oaxaca,Mexico, on July 4, 2002

This paper describes some experiments the author would haveliked to carry out if he had started earlier in the origin-of-life field.The proposal is preceded by a hypothetical outline of the mainevents in the origin of life. According to this outline, the emergence oflife amounts to the transition between two kinds of chemistry: 1) cosmic chemistry, which is beginning to be understood and mostlikely provided the building blocks with which life was first constructed; and 2)biochemistry, the well-known set of enzyme-catalyzedmetabolic reactions that support all living organisms today and must havesupported the universal common ancestor, or LUCA, from which all known formsof life are derived. The pathway leading from one to the other of thosetwo chemistries may be divided into three stages, defined as the pre-RNA, RNA, and protein-DNA stages. A briefsummary of the events that may have occurred in these three stages and ofthe possible underlying mechanisms is given. It is emphasized that theseevents were chemical in nature and, especially, that theymust have prefigured present-day biochemical processes. Protometabolismand metabolism, it is argued, must have been congruent. With congruence as the underlying working hypothesis, threeproblems open to experimental investigation are considered: 1) the involvementof peptides and other multimers as catalysts of early biogenic chemistry;2) the participation of thioesters in primitive energy transactions;and 3) the influence of amino acids on the molecular selection of RNAmolecules.     

Formation of Peptide Bonds from Metastable versus Crystalline Phase: Implications for the Origin of Life

Formation of peptide bonds was attempted bythermal activation of dry amino acids from aqueous solutionthat simulated prebiotic evaporative environments. Theevaporation trend of amino acids solutions shows abifurcation and can lead to either a crystalline phase(near equilibrium) or a metastable non-crystalline phase(far from equilibrium). Only amino acids in this metastablephase are able to form peptide bonds by thermal activationat temperatures that are generated by solar radiationtoday. We suggest that this metastable phase is the idealinitial material to trigger amino acid assemblage withprotein-like structure because provide the driving force(supersaturation) for an intense interaction betweenmonomers of different amino acids and allows activation ofthese monomers in plausible prebiotic conditions.     

A Computer-Glimpse of the Origin of Life

Evolution is assumed to begin in a very particular compartmentalized location with periodic conditions. A highly diversified world is the driving force for the continuous increase in complexity by colonizing increasingly less favourable regions. Modeling the “origin-of-life” a Darwinian cyclic process is simulated (multiplication with sporadic errors followed by a construction and selection). Starting from a RNA-world (R-strands of R₁ and R₂ monomers building Hairpin-Assembler devices) and introducing another kind of monomers (A₁ and A₂ which interlink to the Hairpin-Assembler devices such that they become bound and form an A-oligomer) it is shown that a simple translation apparatus evolves producing enzymes (specific sequences of A₁ and A₂ monomers given by the sequences of R₁ and R₂ monomers on the assembler-strands). Later on D-strands are introduced, which are not capable of participating in the synthesis of A-oligomers. These D-strands become carriers of the genetic information and induce the formation of increasingly complex entities of functionally interplaying components.     

Question 5: On the Chemical Reality of the RNA World

The discovery of catalytic RNA has revolutionised modern molecular biology and bears important implications for the origin of Life research. Catalytic RNA, in particular self-replicating RNA, prompted the hypothesis of an early “RNA world” where RNA molecules played all major roles such information storage and catalysis. The actual role of RNA as primary actor in the origin of life has been under debate for a long time, with a particular emphasis on possible pathways to the prebiotic synthesis of mononucleotides; their polymerization and the possibility of spontaneous emergence of catalytic RNAs synthesised under plausible prebiotic conditions. However, little emphasis has been put on the chemical reality of an RNA world; in particular concerning the chemical constrains that such scenario should have met to be feasible. This paper intends to address those concerns with regard to the achievement of high local RNA molecules concentration and the aetiology of unique sequence under plausible prebiotic conditions. Presented at: International School of Complexity – 4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

Enantiomeric Crystallization from DL-Aspartic and DL-Glutamic Acids: Implications for Biomolecular Chirality in the Origin of Life

Amino acids in living systems consist almost exclusively of the L-enantiomer. How and when this homochiral characteristic of life came to be has been a matter of intense investigation for many years. Among the hypotheses proposed to explain theappearance of chiral homogeneity, the spontaneous resolution of conglomerates seems one of the most plausible. Racemic solids may crystallize from solution either as racemic compounds(both enantiomeric molecules in the same crystal), or lesscommonly as conglomerates (each enantiomer molecule separate indifferent enantiomeric crystals). Only conglomerates can developa spontaneous resolution (one of the enantiomeric molecule crystallizes preferentially, the other one remains in solution).Most of natural amino acids are racemic compounds at moderatetemperatures. How can we expect a hypothetical spontaneous resolution of these amino acids if they are not conglomerates?In this paper we show how DL-aspartic and DL-glutamic amino acids(racemic compounds), crystallize at ambient conditions as trueconglomerates. The experimental conditions here described,that allows this `anomalous' behaviour, could be also found innatural sedimentary environments. We suggest that these experimental procedures and its natural equivalents, have apotential interest for the investigation of the spontaneous resolution of racemic compounds comprising molecules associatedwith the origin of life.     

The Early Phases of Genetic Code Origin: Conjectures on the Evolution of Coded Catalysis

A review of the most significant contributions on the early phases of genetic code origin is presented. After stressing the importance of the key intermediary role played in protein synthesis, by peptidyl-tRNA, which is attributed with a primary function in ancestral catalysis, the general lines leading to the codification of the first amino acids in the genetic code are discussed. This is achieved by means of a model of protoribosome evolution which sees protoribosome as the central organiser of ancestral biosynthesis and the mediator of the encounter between compounds (metabolite-pre-tRNAs) and catalysts (peptidyl-pre-tRNAs). The encounter between peptidyl-pre-tRNA catalysts in protoribosome is favoured by metabolic pre-mRNAs and later resulted (given the high temperature at which this evolution is supposed to have taken place) in the evolution of mRNAs with codons of the type GNS. These mRNAs codified only for those amino acids that the coevolution theory of genetic code origin sees as the precursors of all other amino acids. Some aspects of the model here discussed might be rendered real by the transfer-messenger RNA molecule (tmRNA) which is here considered a molecular fossil of ancestral protein synthesis.     

Origin of amino acid homochirality: relationship with the RNA world and origin of tRNA aminoacylation

The origin of homochirality of l-amino acids has long been a mystery. Aminoacylation of tRNA might have provided chiral selectivity, since it is the first process encountered by amino acids and RNA. An RNA minihelix (progenitor of the modern tRNA) was aminoacylated by an aminoacyl phosphate oligonucleotide that exhibited a clear preference for l- as opposed to d-amino acids. A mirror-image RNA system with l-ribose exhibited the opposite selectivity, i.e., it exhibited an apparent preference for the d-amino acid. The selectivity for l-amino acids is based on the stereochemistry of RNA. The side chain of d-amino acids is located much closer to the terminal adenosine of the minihelix, causing them collide and interfere during the amino acid-transfer step. These results suggest that the putative RNA world that preceded the protein theatre determined the homochirality of l-amino acids through tRNA aminoacylation.     

Selective labeling and detection of specific RNAs in an RNA mixture

We report here a unique approach to selectively label and detect specific RNA in an RNA mixture (without separation or purification) using DNA polymerase, dNTP labels, and a short synthetic DNA template complementary to the 3(')-terminus of the RNA. The detection sensitivity is high, at attomole level (10-18 mole). The selective principle was demonstrated by individually labeling and detecting RNAs in a RNA mixture when different templates were provided. By taking advantage of the template-directed selectivity, poly(A) tail-containing mRNA in total RNA was detected and labeled at the 3(')-terminal on a poly(T) template. Nonradioactive labels, such as fluorophore and antigen labels, may also be used; this method can be applied in methodology for direct detection and quantification of viral RNAs.