The origin of life: chemical evolution of a metabolic system in a mineral honeycomb?

For the RNA-world hypothesis to be ecologically feasible, selection mechanisms acting on replicator communities need to be invoked and the corresponding scenarios of molecular evolution specified. Complementing our previous models of chemical evolution on mineral surfaces, in which selection was the consequence of the limited mobility of macromolecules attached to the surface, here we offer an alternative realization of prebiotic group-level selection: the physical encapsulation of local replicator communities into the pores of the mineral substrate. Based on cellular automaton simulations we argue that the effect of group selection in a mineral honeycomb could have been efficient enough to keep prebiotic ribozymes of different specificities and replication rates coexistent, and their metabolic cooperation protected from extensive molecular parasitism. We suggest that mutants of the mild parasites persistent in the metabolic system can acquire useful functions such as replicase activity or the production of membrane components, thus opening the way for the evolution of the first autonomous protocells on Earth.

The basal phylogenetic position of Nanoarchaeum equitans (Nanoarchaeota).

Using sequences of ribosomal RNA from organisms belonging exclusively to the Archaea domain and by means of two methods to remove the phylogenetic noise, we investigate the phylogenetic position of Nanoarchaeum equitans. The results obtained are compatible with the hypothesis that N. equitans represents a new phylum within the Archaea domain because the characteristic long branch of N. equitans in phylogenetic trees is conserved even after most of the phylogenetic noise has been removed, thus implying that its rRNA might indeed be singular. However, our analysis is unable to be equally as clear on the phylogenetic position of Methanopyrus kandleri.

Montmorillonite protection of an UV-irradiated hairpin ribozyme: evolution of the RNA world in a mineral environment.

BACKGROUND: The hypothesis of an RNA-based origin of life, known as the "RNA world", is strongly affected by the hostile environmental conditions probably present in the early Earth. In particular, strong UV and X-ray radiations could have been a major obstacle to the formation and evolution of the first biomolecules. In 1951, J. D. Bernal first proposed that clay minerals could have served as the sites of accumulation and protection from degradation of the first biopolymers, providing the right physical setting for the evolution of more complex systems. Numerous subsequent experimental studies have reinforced this hypothesis. RESULTS: The ability of the possibly widespread prebiotic, clay mineral montmorillonite to protect the catalytic RNA molecule ADHR1 (Adenine Dependent Hairpin Ribozyme 1) from UV-induced damages was experimentally checked. In particular, the self-cleavage reaction of the ribozyme was evaluated after UV-irradiation of the molecule in the absence or presence of clay particles. Results obtained showed a three-fold retention of the self-cleavage activity of the montmorillonite-protected molecule, with respect to the same reaction performed by the ribozyme irradiated in the absence of the clay. CONCLUSION: These results provide a suggestion with which RNA, or RNA-like molecules, could have overcame the problem of protection from UV irradiation in the RNA world era, and suggest that a clay-rich environment could have favoured not only the formation of first genetic molecules, but also their evolution towards increasingly complex molecular organization.

Catalytic activity of hammerhead ribozymes in a clay mineral environment: implications for the RNA world.

The hypothesized RNA-based world would have required the presence of a protected environment in which RNA, or an RNA-like molecule, could originate and express its biological activity. Recent studies have indicated that RNA molecules adsorbed/bound on clay minerals are able to persist in the presence of degrading agents, to interact with surrounding molecules, and to transmit the information contained in their nucleotide sequences. In this study, we assessed the ability of RNA molecules with catalytic activity to perform a specific reaction in a mineral environment. For this purpose, we investigated the self-cleavage reaction of the hammerhead ribozyme of the Avocado Sun Blotch Viroid (ASBVd), both in the monomeric and in dimeric forms. The monomeric transcript was tightly bound on the clay mineral montmorillonite to form a stable complex, while the behaviour of the dimeric transcript was studied in the presence of the clay particles in the reaction mixture. The results indicated that the hammerhead ribozyme was still active when the monomeric transcript was adsorbed on the clay surface, even though its efficiency was reduced to about 20% of that in solution. Moreover, the self-cleavage of clay-adsorbed molecule was significantly enhanced ( approximately four times) by the presence of the 5' reaction product. The self-cleavage reaction of the dimeric transcript in the presence of montmorillonite indicated that the mineral particles protected the RNA molecules against aspecific degradation and increased the rate of cleavage kinetics by about one order of magnitude. These findings corroborate the hypothesis that clay-rich environments would have been a good habitat in which RNA or RNA-like molecules could originate, accumulate and undergo Darwinian evolutionary processes, leading to the first living cells on Earth.

The first lines of divergence in the Bacteria domain were the hyperthermophilic organisms, the Thermotogales and the Aquificales, and not the mesophilic Planctomycetales.

In order to establish whether the first lines of divergence in the Bacteria domain were the mesophilic or the hyperthermophilic organisms, we have performed a phylogenetic analysis on a concatenamer obtained from the fusion of 20 different proteins. The phylogenetic analysis carried out using five different methods has shown that, contrary to what is reported in the literature [Brochier, C., Philippe, H., 2002. A non-hyperthermophilic ancestor for Bacteria. Nature 417, 244], it was probably the hyperthermophilic organisms, the Thermotogales and the Aquificales, which were the first lines of divergence in the Bacteria domain, and not the mesophilic Planctomycetales. This strengthens the hypothesis that the last universal common ancestor might have been a hyperthermophilic 'organism' and that, more generally, life might have originated at high temperature.

Investigation of de novo totally random biosequences, Part III: RNA Foster: A novel assay to investigate RNA folding structural properties.

Fold is essential to RNA properties, and, in particular, its thermodynamic stability can be used to monitor RNA-protein or RNA-ligand interactions, and to engineer RNA with novel or improved properties. While clearly valuable, experimental determination of RNA folding stability by traditional biophysical techniques requires substantial amounts of pure sample and rather expensive equipment. In this paper, we report a new, simple approach to the determination of RNA folding stability by coupling enzymatic digestion and temperature denaturation. The assay, named RNA folding stability Test (RNA Foster), is designed to probe the fraction of folded RNA (f(fold)) in an equilibrium mixture of folded and unfolded ones as a function of temperature. The simplicity of RNA Foster suggests that it can easily be scaled up for high-throughput studies of RNA folding stability both in basic and applied research.

Investigation of de novo totally random biosequences, Part IV: Folding Properties of de novo, totally random RNAs.

This work lies within the framework of a broader project aimed at exploring the realm of all possible folded polypeptides; the main question addressed here is whether the corresponding RNAs also assume a folded conformation. We present an investigation on the structural properties of de novo, totally random RNAs by means of the 'RNA Foster' assay. Experimental results show that all RNAs studied are folded at 37 degrees , so that fold seems to be a common feature of RNAs. Random RNAs' fold shows a surprising thermal stability with an average T(m) value at ca. 50 degrees which prompts the idea that thermo-stable structures might not be as rare as they are commonly thought to be. The results are discussed within the general framework of random RNA properties such as those that might have been produced in a prebiotic scenario.

A surface-mediated origin of the RNA world: biogenic activities of clay-adsorbed RNA molecules.

The involvement of clay surfaces in the origin of the first genetic molecules on Earth has long been suggested. However, the formation of these polymers was not sufficient by itself to initiate the evolutionary process leading to the appearance of life. These macromolecules had to persist in primeval habitats so that their biological potentiality could be expressed. In this study, we assess the possibility of development of the RNA world on a clay substrate by investigating the capacity of different RNA molecules adsorbed/bound on the clay minerals montmorillonite (M) and kaolinite (K) to persist in the presence of a degrading agent (RNase-A), to interact specifically with complementary RNA strands, and to transmit the information contained in their nucleotide sequences. The RNase-A degradation of clay-adsorbed 23S rRNA from Escherichia coli was significantly slower (75-80%) than that observed for free rRNA, and the complete digestion of nucleic acid in the presence of clay was obtained in 2 vs. 1 h. Clay-adsorbed Poly[A] homopolymer was able to recognize the complementary Poly[U] homopolymer present in the surrounding water solution and to establish a specific interaction (association) with it, possibly leading to the formation of double strands. Reverse transcription and amplification (RT-PCR) amplification of free and clay-adsorbed 16S indicated that the presence of clay particles partially reduced the efficiency and processivity of reverse transcriptase but did not inhibit its activity, demonstrating that clay-adsorbed RNA is still available for enzymatic replication. These findings indicate that primordial genetic molecules adsorbed on clay minerals would have been protected against degrading agents present in the environment and would have been in the right conditions to undergo evolutionary processes.

Carbonic anhydrase inhibitors. Inhibition of mitochondrial isozyme V with aromatic and heterocyclic sulfonamides.

The first inhibition study of the mitochondrial isozyme carbonic anhydrase (CA) V (of murine origin) with a series of aromatic and heterocyclic sulfonamides is reported. Inhibition data of the cytosolic isozymes CA I and CA II and the membrane-bound isozyme CA IV with these inhibitors are also provided for comparison. Several low nanomolar CA V inhibitors were detected (KI values in the range of 4-15 nM), most of them belonging to the acylated sulfanilamide, ureido-benzenesulfonamide, 1,3,4-thiadiazole-2-sulfonamide, and aminobenzolamide type of compounds. The clinically used inhibitors acetazolamide, methazolamide, ethoxzolamide, dorzolamide, brinzolamide, and topiramate on the other hand were less effective CA V inhibitors, showing inhibition constants in the range of 47-63 nM. Some of the investigated sulfonamides, such as the ureido-benzenesulfonamides and the acylated sulfanilamides showed higher affinity for CA V than for the other isozymes, CA II included, which is a remarkable result, since most compounds investigated up to now inhibited the cytosolic isozyme CA II better. These results prompt us to hypothesize that the selective inhibition of CA V, or the dual inhibition of CA II and CA V, may lead to the development of novel pharmacological applications for such sulfonamides, for example in the treatment or prevention of obesity, by inhibiting CA-mediated lipogenetic processes.

Ribozymes: Flexible molecular devices at work.

The discovery of ribozymes, RNAs with catalytic activity, revealed the extraordinary characteristic of this molecule, and corroborated the idea that RNA was the first informative polymer. The "RNA world" hypothesis asserts that the DNA/RNA/PROTEIN world arose from an earlier RNA world in which were present only RNA molecules able to perform both of the two functions performed separately by DNA and proteins in the present-day cells: the ability to transfer genetic information and to carry out catalytic activity. The catalytic properties of ribozymes are exclusively due to the capacity of RNA molecules to assume particular structures. Moreover, the structural versatility of RNA can allow to a single RNA sequence to fold in more than one structure, able to perform more than one function. In the first part of this work we will discuss the RNA plasticity, focusing on "bifunctional" ribozymes isolated by in vitro selection experiments, and on the consequences of this plasticity in the prospective of the emergence of new specific functions. The possibility that one sequence could have more than one structure/function, greatly increase the evolutionary potential of RNA, and the capacity of RNA to switch from a structure/function to another is probably one of the reasons of the evolutionary success also in modern-day cells. Naturally occurring ribozymes discovered in contemporary cells, demonstrate the crucial role that ribozymes still have in the modern protein world. In the second part of this paper we will discuss the capacity of natural ribozymes to modulate gene expression making use of their exclusive catalytic properties. Moreover, we will consider the possibility of their ancient origin.

Natural and unnatural ribozymes: back to the primordial RNA world.

We review natural and in vitro selected ribozymes, for which combined studies could provide us with both insight into the functions performed by ancient RNA molecules in a primitive RNA world and a hypothesis about evolutionary steps that led to the contemporary world.

Looking for the primordial genetic honeycomb.

All life forms on Earth share the same biological program based on the DNA/RNA genomes and proteins. The genetic information, recorded in the nucleotide sequence of the DNA and RNA molecule, supplies the language of life which is transferred through the different generations, thus ensuring the perpetuation of genetic information on Earth. The presence of a genetic system is absolutely essential to life. Thus, the appearance in an ancestral era of a nucleic acid-like polymer able to undergo Darwinian evolution indicates the beginning of life on our planet. The building of primordial genetic molecules, whatever they were, required the presence of a protected environment, allowing the synthesis and concentration of precursors (nucleotides), their joining into larger molecules (polynucleotides), the protection of forming polymers against degradation (i.e. by cosmic and UV radiation), thus ensuring their persistence in a changing environment, and the expression of the "biological" potential of the molecule (its capacity to self-replicate and evolve). Determining how these steps occurred and how the primordial genetic molecules originated on Earth is a very difficult problem that still must be resolved. It has long been proposed that surface chemistry, i.e. on clay minerals, could have played a crucial role in the prebiotic formation of molecules basic to life. In the present work, we discuss results obtained in different fields that strengthen the hypothesis of a clay-surface-mediated origin of genetic material.

Molecular recognition of metal complexes by DNA: a comparative study of the interactions of the parent complexes [PtCl(TERPY)]Cl and [AuCl(TERPY)]Cl2 with double stranded DNA.

The interactions of the parent complexes [AuCl(Terpy)]Cl(2) and [PtCl(Terpy)]Cl with DNA were analysed by various physicochemical methods. Surprisingly, these metal complexes produce different interaction patterns with DNA in spite of their profound structural similarity. Indeed, important modifications are detected in the characteristic UV-Vis bands of [PtCl(Terpy)]Cl upon addition of ct-DNA, while the spectrum of [AuCl(Terpy)]Cl(2) is almost unaffected. Gel electrophoresis studies confirm these findings: [PtCl(Terpy)]Cl - but not [AuCl(Terpy)]Cl(2) - retards significantly the mobility of the supercoiled form of the pHV14 plasmid after a short incubation time. Ultrafiltration studies indicate that the affinity of [PtCl(Terpy)]Cl for ct-DNA is significantly greater than that of [AuCl(Terpy)]Cl(2). On the other hand, both [AuCl(Terpy)]Cl(2) and [PtCl(Terpy)]Cl induce important changes in the CD spectrum of ct-DNA, at high concentration, and increase its T(m) value. Remarkably, the analysed metal-complex/DNA interaction patterns depend critically on the incubation times. We propose that [PtCl(Terpy)]Cl quickly intercalates DNA; then, formation of coordinative bonds progressively takes place with time. At variance, [AuCl(Terpy)]Cl(2) first interacts electrostatically with the DNA surface, with subsequent slow formation of some coordinative bonds.

Origin, persistence and biological activity of genetic material in prebiotic habitats.

Molecules which store genetic information (i.e. RNA and DNA) are central to all life on Earth. The formation of these complex molecules, and ultimately life, required specific conditions, including the synthesis and concentration of precursors (nucleotides), the joining of these monomers into larger molecules (polynucleotides), their protection in critical conditions (like those probably existing in primeval habitats), and the expression of the biological potential of the informational molecule (its capacity to multiply and evolve). Determining how these steps occurred and how the earliest genetic molecules originated on Earth is a problem that is far from being resolved. Recent observations on the polymerization of nucleotides on clay surfaces and on the resistance of clay-adsorbed nucleic acids to environmental degradation suggest that clay minerals could have acted as a resting place for the formation and preservation of prebiotic genetic molecules, whatever they were, and for the self-organization of the first auto-replicating systems. In the present work, the molecular characteristics and biological activity of different nucleic acids (DNA, RNAs) adsorbed/bound on clay minerals are discussed in the light of their possible role in ancestral environments.

Cations as mediators of the adsorption of nucleic acids on clay surfaces in prebiotic environments.

Monovalent ([Na+] > 10 mM) and divalent ([Ca2+], [Mg2+] > 1.0 mM) cations induced the precipitation of nucleic acid molecules. In the presence of clay minerals (montmorillonite and kaolinite), there was adsorption instead of precipitation. The cation concentration needed for adsorption depended on both the valence of the cations and the chemical nature of the nucleic acid molecules. Double-stranded nucleic acids needed higher cation concentrations than single-stranded ones to be adsorbed to the same extent on clay. Divalent cations were more efficient than monovalent ones in mediating adsorption. Adsorption to the clay occurred only when both nucleic acids and cations were present. However, once the complexes were formed, the cations could not be removed from the system by washing, indicating that they are directly involved in the association between nucleic acids and mineral surfaces. These observations indicate that cations take part directly in the formation of nucleic acid-clay complexes, acting as a 'bridge' between the negative charges on the mineral surface and those of the phosphate groups of the genetic polymer. The relatively low cation concentrations needed for adsorption and the ubiquitous presence of clay minerals in the environment suggest that the adsorption of nucleic acids on mineral surfaces could have taken place in prebiotic habitats. This may have played an important role in the formation and preservation of nucleic acids and/or their precursor polymers.

Carbonic anhydrase inhibitors. inhibition of cytosolic isozymes I and II and transmembrane, cancer-associated isozyme IX with anions.

Except for sulfonamides, metal complexing anions represent the second class of inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). The first inhibition study of the transmembrane, tumor-associated isozyme CA IX with anions is reported here. Inhibition data of the cytosolic isozymes CA I and CA II with a large number of anionic species such as halides, pseudohalides, bicarbonate, nitrate, hydrosulfide, arsenate, etc., are also provided for comparison. Isozyme IX has an inhibition profile by anions different in some aspects from those of CA I and CA II, that may have interesting physiological consequences.

Carbonic anhydrase inhibitors: inhibition of the tumor-associated isozyme IX with aromatic and heterocyclic sulfonamides.

The inhibition of the tumor-associated transmembrane carbonic anhydrase IX (CA IX) isozyme has been investigated with a series of aromatic and heterocyclic sulfonamides, including the six clinically used derivatives acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide and brinzolamide. Inhibition data for the physiologically relevant isozymes I and II (cytosolic forms) and IV (membrane-bound) were also provided for comparison. A very interesting and unusual inhibition profile against CA IX with these sulfonamides has been observed. Several nanomolar (K(I)-s in the range of 14-50 nM) CA IX inhibitors have been detected, both among the aromatic (such as orthanilamide, homosulfonilamide, 4-carboxy-benzenesulfonamide, 1-naphthalenesulfonamide and 1,3-benzenedisulfonamide derivatives) as well as the heterocylic (such as 1,3,4-thiadizole-2-sulfonamide, etc.) sulfonamides examined. Because CA IX is a highly active isozyme predominantly expressed in tumor tissues with poor prognosis of disease progression, this finding is very promising for the potential design of CA IX-specific inhibitors with applications as anti-tumor agents.

Carbonic anhydrase inhibitors. Inhibition of cytosolic isozymes I and II and transmembrane, cancer-associated isozyme IX with lipophilic sulfonamides.

A series of new compounds was obtained by reaction of aromatic/heterocyclic sulfonamides incorporating amino groups with N,N-diphenylcarbamoyl chloride and diphenylacetyl chloride. These sulfonamides were assayed for the inhibition of three carbonic anhydrase (CA, EC 4.2.1.1) isozymes: the cytosolic CA I and CA II, and the transmembrane, cancer-associated isozyme CA IX. Good inhibitors against all these isoforms were detected, and the inhibition profile of the newly investigated isozyme IX was observed to be different from that of the cytosolic isozymes, I and II. This may lead to the development of novel anticancer therapies based on the selective inhibition of CA IX.

Carbonic anhydrase inhibitors: inhibition of human and murine mitochondrial isozymes V with anions.

In addition to sulfonamides, metal complexing anions represent the second class of inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). The first inhibition study of the mitochondrial isozyme CA V (of murine and human origin) with anions is reported here. Inhibition data of the cytosolic isozymes CA I and CA II as well as the membrane-bound isozyme CA IV with a large number of anionic species such as halides, pseudohalides, bicarbonate, nitrate, hydrosulfide, arsenate, sulfamate, and sulfamidate and so on, are also provided for comparison. Isozyme V has an inhibition profile by anions completely different to those of CA I and IV, but similar to that of hCA II, which may have interesting physiological consequences. Similarly to hCA II, the mitochondrial isozymes show micro-nanomolar affinity for sulfonamides such as sulfanilamide and acetazolamide.