RNA Constituents

Prebiotic evolution, as concluded by Orgel, in all likelihood went through an RNA World:

"The demonstration that ribosomalpeptidesynthesis is a ribozyme-catalyzed reaction makes it almost certain that there was once an RNA World."6

Accordingly extensive research has been directed to the plausible abiotic syntheses of the constituents of RNA.

(i) Nucleobases

The synthesis ofadenine from hydrogen cyanide by Oro initiated the prebiotic chemistry of nucleic acids.22 Refluxing of ammonium cyanide forms the HCN tetramer, which is further polymerized to a dark intractable solid from which adenine and guanine can be recovered.6 Tetramer formation requires HCN concentrations higher than 10 mM. Since it would not be possible to reach such a concentration in the bulk oceans and concentration via evaporation also could not work with as volatile a molecule as HCN, a more plausible method for concentrating HCN is eutectic freezing: if a dilute aqueous solution of HCN is cooled below 0°C, pure ice crystallizes out and the solution becomes more concentrated until a eutectic point is reached at -23.4°C at 74.5 (moles)% which gives significant yields of adenine.23,24 There are also HCN-independent routes of purine synthesis.25 The feasibility of prebiotic synthesis of purines is confirmed by their occurrence on the Murchison meteorite (Table 6.2).

Cyanoacetylene is a major product formed when electric discharge is passed through a mixture ofnitrogen and methane. It reacts with two molecules of cyanic acid to give cytosine. It also can be hydrolysed to cyanoacetaldehyde, which condenses with urea to give cytosine (Fig. 9.2). Hydrolysis of cytosine yields uracil.6,26,27 Since cytosine is thermally unstable with a t1/2 ofonly 17,000 years even at 0°C,28,29 it would have to be replenished continually, or else utilized for RNA or RNA-like biopolymers only at a later stage.

(ii) Nucleosides

The polymerization of formaldehyde in the presence of simple mineral catalysts to form a mixture of sugars is known as the formose reaction. Discovered by Butlerow in the 19th Century, it is a unique cyclic autocatalytic process that converts the simple four-atom formaldedye, found even in interstellar gas (Table 9.1), into a complex mixture of sugars. Since the complexity of the formose reaction products results in a low fraction of ribose among the products, doubts have been voiced regarding the plausibility of prebiotic ribose synthesis by means of this reaction. However, in the presence of Pb+2 ion and catalytic amounts of an intermediate in the pentose pathway, pentoses including ribose can exceed 30% of reaction products, thus furnishing prebiotic ribose.30

Alkaline environments, favorable to the formose reaction but once thought to be rare, could be found in alkaline hydrothermal systems. This together with borate stabilization of ribose further enhance the probability of ribose production on primitive Earth.31 As well, ribose permeates fatty acid and phospholipid membranes more rapidly than other 5- or 6-carbon aldo-sugars, which might be a significant factor favoring its selection as an RNA constituent.32 The carbonaceous Murchison and Murray meteorites contain a variety of polyols including glycolic acid, glyceric acid, dihy-droxyacetone, glycerol, erythritol, threitol and ribitol which can be converted into sugars. These meteoritic components, besides testifying to the feasibility of abiotic synthsis of polyols under prebiotic conditions, could furnish polyols to the primitive life forms for nucleic acid synthesis and intermediary metabolism.33

When D-ribose is heated with hypoxanthine in the presence of magnesium chloride or sea salts, up to 8% P-D-inosine is formed. With adenine, reaction with D-ribose under similar conditions followed by hydrolysis likewise yields up to 3% P-D-adenosine. No direct synthesis of pyrimidine nucleosides from ribose and uracil or cytosine has been reported, but a-cytidine may be obtained from reaction of ribose or ribose phosphate with cyanamide and cyanoacetylene in aqueous solution and photo-converted to P-cytidine.6,34,35 Overall, the formation of nucleosides represents at present one of the weakest links in the chain of prebiotic reactions leading to oliogonucleotides.6

(iii) Nucleotides

In modern organisms, nucleosides are phosphorylated by kinases to form nucleotides using ATP as phosphorylating agent. The attractive properties of phosphate in energy transfers stem from the ability of phosphorus to form multiple bonds of moderate strength and the tribasic nature of phosphoric acid. Inorganic polyphosphates (polyP) such as trimetaphosphate and linear polyphosphates and to some extent pyrophosphate, could function as prebiotic phosphorylating agents for nucleotide synthesis. Although the concentration of inorganic phosphate in the hydrosphere is expected to be as low as 0.1 nM owing to the low solubility of calcium phosphate at neutral pH, an adequate supply of inorganic phosphate might be acquired via a number of routes including:36

a. Although many common phosphorus-containing minerals aside from schreibersite from meteorites are only slightly soluble in water, it is estimated that up to 5% oftotal crustal P on early Earth might be extraterrestrial schreibersite added to the Earth by meteoritic impacts. Fe3P, a model for schreibersite, reacts with water to yield orthophosphate and condensed phosphates.

b. Decreased alkalinity leads to the precipitation of the acid calcium salt brushite (CaHPO4.2H2O) and high magnesium and ammonia concentrations favor the precipitation ofstruvite (MgNH4PO4.6H2O). Both brushite and struvite can form condensed phosphate upon heating.

c. Under strongly reducing conditions, phosphate is reduced to phosphite. Ammonium phosphite reacts with nucleo-sides to yield nucleoside-phosphites, which in the dimeric form is easily oxidized to a phosphodiester bond.

d. Pyrophosphate and polyP have been obtained from volcanic fumaroles.37

Nucleosides can be converted, not very efficiently, to nucleotides by heating in the solid state with acidic phosphates.38 Heating ammonium phosphate with urea yields a mixture of linear phosphates in the absence ofan organic component and nucleotides in excellent yields in the presence of nucleosides. For example, when uridine is heated with excess urea and ammonium phosphate at 100°C, 70% of the uridine is converted to a complex mixture of uridine nucleotides, with some possibility ofstructuring the reaction conditions to favor a particular form of uridine phosphate.6 Reaction of trimetaphosphate with adensosine catalyzed by magnesium gives about 9% nucleotides, mainly 2',3'-cyclic AMP, at neutral pH.39 Introduction ofwet-dry cycles and catalysis by Ni(II) enhanced the yield of nucleotides to 30%, producing up to 10% 2'3'-cyclic AMP as well as 13% ATP.40 These studies point to the prebiotic availability of polyP, ATP as high-energy compounds and NTPs as substrates for RNA polymerization.

(iv) Non-Canonical Backbones

D-ribose is utilized in the construction ofboth cellular and viral RNA, suggesting that the use of this particular pentose has been a long standing one. Nonetheless, it does not necessarily follow that other forms of nucleobase-containing biopolymers might not have preceded RNA. In fact, a range ofalternative nucleobase-containing biopolymers have been explored with respect to their potential as predecessor replicators/genes prior to the advent ofthe RNA World based on a canonical backbone where phosphate is joined to the 3'-OH and 5'-OH of adjacent ribose-furanose residues:

a. Owing to the relative difficulty ofabiotic synthesis ofnucle-osides and the occurrence of enantiomeric cross-inhibition, it was suggested that ribose in the RNA backbone might be advantageously replaced by acyclic analogues derived from glycerol, acrolein or erythritol in order to overcome such adverse effects.41

b. Pyranosyl-RNA (p-RNA), with a backbone of phosphate joined to the 2'-OH and 4'-OH ofadjacent ribose-pyranose residues, has a stronger and more selective base-pairing system than RNA.42,43





n=c-ch2-cho +







c. Threofuranosyl nucleic acid (TNA) has a backbone where the five-carbon ribose is replaced by the four-carbon threose (Fig. 9.3). It forms stable heteroduplexes with both RNA and DNA.44 One disadvantage with TNA is the absence of free OH-group from its backbone, which would reduce its capability to function as threozymes and explain the thorough displacement of what prebiotic TNA there was by RNA.

d. Peptide nucleic acids (PNA) contain the usual nucleobases and pair with nucleic acids, but are free ofphosphate.45 PNA has been considered as a prebiotic biopolymer,46 but so far no straightforward prebiotic synthesis of PNA monomers has been reported.6 Moreover, the lower global flexibility ofPNA relative to RNA47 suggests that PNA might experience greater difficulty than RNA in developing small-sized aptamers or PNA-zymes.

These alternatives serve the valuable purpose of focusing attention on the possibility of RNA replicators being preceded by other informational biopolymers in prebiotic evolution. TNA offers the advantage that its ability to form a TNA-RNA duplex might allow transition of an early usage of TNA, or co-usage of TNA and RNA, to a subsequent RNA World with limited disruption of the replicator system.

Nowadays, organisms employ DNA genes, whereas viruses employ both RNA and DNA genes. Evidence suggesting that DNA appeared after RNA48 and even after proteins,49 includes:

a. Greater stability of the phosphodiester backbone of DNA compared to RNA;

b. Absence of proofreading by RNA polymerases leading to higher mutation rates in RNA genomes;

c. Information in RNA degrades because deamination ofcyto -sine to form uracil can be detected and repaired in DNA but not in RNA, thereby leading to higher mutation and error rates in RNA genomes compared to DNA genomes;

d. UV irradiation produces more photochemical changes in RNA than in DNA;

e. Deoxyribonucleotides are formed from reduction of ribonucleotides by ribonucleotide reductase;

f. Use of a free radical in the catalytic mechanism of ribonucleotide reductase that likely could be fashioned only by a sophisticated protein.

Accordingly, the evolution of genes likely began with RNA, TNA or some other predecessor replicators and evolved to an RNA World. Subsequently the RNA genes were replaced by DNA in cells, while viruses can choose between DNA (e.g., smallpox virus) or RNA (e.g., tobacco mosaic virus) genes. The primitive replicators had to either act as biocatalysts themselves or encode the formation of biocatalysts, which imposes important constraints on the nature of the first replicators. RNA meets the biocatalytic requirement through its ribozymic activities (Sections 11.2). Proteins are virtuoso biocatalysts but cannot readily self-replicate. One expects some biocatalytic capability from pRNA as 'p-ribozyme', TNA as 'threozyme' and PNA as 'PNzyme', but it remains to be determined just how well they perform in these capacities.

9.4 Lipids

Amphiphiles are compounds containing a hydrophilic head group that is attracted to water and a hydrophobic tail that is attracted to organic solvents immiscible with water. Lipoidal substances are bioamphiphiles consisting oflong chain fatty acids or fatty alcohols where a carboxylate or alcohol moiety furnishes the hydrophilic head group of the elongated molecule and a long alkane chain the hydrophobic tail; or phospholipids where a phosphate-moiety furnishes the hydrophilic head group and alkane chains the hydrophobic tail. When placed in water, amphiphilic molecules tend to arrange themselves into micelles with their tails clustered together inside the micelle away from the water molecules they dislike and their heads orientated toward the surrounding water molecules to which they are attracted. The amphiphiles may also self-assemble into vesicles with a bilayer membrane, where two layers of the am-phiphile are aligned with their tails toward one another and their heads facing water on either side of the membrane.

When samples of the Murchison meteorite were extracted with organic solvent and allowed to interact with an aqueous phase, self-assembled membrane vesicles were observed. This discovery strikingly points to the existence of extraterrestrial amphiphiles and the ease with which they can generate membrane vesicles. The prebiotic availability ofamphiphiles, their self-assembly into vesicles and the capacity of such vesicles to grow and divide (Section 12.4) facilitated the development of vesicular life forms.50

Amphiphiles might also be produced at submarine hydrothermal vents and deep subterranean hot aquifers, where thermal energy, mineral surfaces and a reducing hydrogen-carbon dioxide gaseous composition promote the synthesis ofa variety oforganic compounds (Section 1.4). In a model prebiotic reaction using oxalic acid as starting material, aqueous Fischer-Tropsch type reaction at hydrothermal temperatures gave rise to more than 5% lipids. Both n-alkanols, which are

employed in archaeal lipids, and n-alkanoic acids, which are employed in bacterial and eukaryotic lipids, were obtained.51

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