RNA Monomers

Although the synthesis of the different RNA monomers from prebiotic molecules is mostly beyond the scope of this chapter (see ref. 2), a quick summary ofresearch efforts on this topic is necessary. The implication of monomer syntheses (or the lack thereof) for the de novo emergence of an RNA World from prebiotic organic mixtures is essential for our discussion.

(i) Synthesis ofthe Ribonucleotides

RNA monomers, P-D-ribonucleotides, are rather complex molecules formed by three different types of molecules—a nucleobase, a ribose and a phosphate group (see Box 10.1), each ofwhich needs to be synthesized as an intermediate product (or made available in the case of the phosphate group) before being assembled into the ribonucleotide.

Experiments aiming at prebiotic nucleotide synthesis have generally concentrated on one of these specific moieties:

• They have demonstrated the synthetic pathways for the formation of both purines and pyrimidines of interest among a large number of nonbiological derivatives.

• D-ribose can be generated by the formose reaction from a prebiotic substrate, formaldehyde, but ribose represents only a rather small fraction of all sugar products. In addition, this sugar decomposes with its half-life being between hours and years at pH 7, depending on the temperature. This situation can however be partially remedied either by complexation with borate or derivatization of the ribose.

• Nucleoside synthesis is obtained by coupling a ribose with a nucleobase and currently represents the weakest step in prebiotic nucleotide synthesis. Recent studies however hint at a simplified synthesis when starting with a ribose derivatized by phosphate.10

• The nucleoside can react with inorganic phosphate to yield nucleotide 5'-phosphates.

Although these "prebiotically plausible" syntheses are conducted with highly purified reactants, they still deliver low yields for any given product. Multiple steps such as solvent extractions are involved, which are certainly not prebiotic. Finally, they produce a complex mixture ofcompounds, some ofwhich are isomers (molecules with the same chemical formula and often with the same kinds of bonds between atoms, but in which the atoms are arranged differently) of the intended products and which could easily inhibit polymerization or replication.

The difficulties encountered in the syntheses are so severe that alternatives to the de novo appearance of RNA on the primitive Earth should be seriously considered. For example, (i) the nucle-

Activity i Conformation i

Secondary structures motifs Tertiary structure motifs

Nucleobase sequence

Figure 10.2. Chain of dependence for RNA activity. The catalytic activity of an RNA depends on its conformation that is obtained from the interactions between secondary and tertiary structure motifs. In turn, the assembly of such motifs is conditioned by the nucleobase sequence.

Box 10.1. Formation of RNA Structures

RNA strands can form a variety of structures that are integral to the in vivo functions of the molecules. RNA structures likely played a significant role in the RNA World as they do for in-vitro selected ribozymes today.

The structures will mainly depend on the RNA molecule sequences and their base-pairing behavior, that is, their capacity to form hydrogen bonds (H-bonds) between two nucleobases, as well as unspecific nucleobase stacking. Other properties such as the torsion angles along the phosphate-ribose backbone (Fig. Box 10.1.1A), the nucleobase and ribose conformations (Fig. Box 10.1.1B) will also contribute to structure formation mainly by constraining the molecular conformations (see ref. 1). The various base pairs are classified into two categories: the common Watson-Crick base pairs (Fig. Box 10.1.1C) and the nonWatson-Crick base pairs (Fig. Box 10.1.1D).

Figure Box 10.1.1. Factors influencing RNA structures. A) RNA polymer is formed of |3-D-ribonucleotides linked by phosphodiester linkages. Each nucleotide is composed of a backbone consisting of phosphate (dark highlighted)-ribose (light highlighted) group and a nucleobase, a heterocycle, which is attached to the C1'ofthe ribose in a | configuration (both nucleobase and phosphate above the ribose-ring plane). Nucleobases are classified as purines, e.g., Adenine (A) and Guanine (G), or pyrimi-dines, e.g., Cytosine (C) and Uridine (U). Each single bond (dotted fat lines) along its backbone can rotate. B) The D-ribose can adopt two types of conformations (or puckering forms) C2- and C3-endos. C) Watson-Crick base pairs: A^U and G^C interact via two and three hydrogen bonds (dotted lines), respectively. An N-H group acts as hydrogen donor, while a nonbinding electron pair of an oxygen on the nucleobase ring (C = O) or a ring nitrogen are the acceptors. D) Non-Watson-Crick base pairs; on the left, a Wobble base-pair system (G^U); and, on the right, some additional nonWatson-Crick base pairs that occur in RNA.

Figure Box 10.1.1. Factors influencing RNA structures. A) RNA polymer is formed of |3-D-ribonucleotides linked by phosphodiester linkages. Each nucleotide is composed of a backbone consisting of phosphate (dark highlighted)-ribose (light highlighted) group and a nucleobase, a heterocycle, which is attached to the C1'ofthe ribose in a | configuration (both nucleobase and phosphate above the ribose-ring plane). Nucleobases are classified as purines, e.g., Adenine (A) and Guanine (G), or pyrimi-dines, e.g., Cytosine (C) and Uridine (U). Each single bond (dotted fat lines) along its backbone can rotate. B) The D-ribose can adopt two types of conformations (or puckering forms) C2- and C3-endos. C) Watson-Crick base pairs: A^U and G^C interact via two and three hydrogen bonds (dotted lines), respectively. An N-H group acts as hydrogen donor, while a nonbinding electron pair of an oxygen on the nucleobase ring (C = O) or a ring nitrogen are the acceptors. D) Non-Watson-Crick base pairs; on the left, a Wobble base-pair system (G^U); and, on the right, some additional nonWatson-Crick base pairs that occur in RNA.

Structures stabilized by interactions between two strands or by binding of metal ions or proteins on the RNA are classified into categories of secondary and tertiary structure motifs (Fig. Box 10.1.2).

Hairpins are the most common motif in RNA folding. They are composed of a generally short, double-stranded, helical stem and a terminal loop of variable numbers of nucleotides. They clearly illustrate the relation between nucleobase sequence and strand conformation that allows the stabilization of an RNA fold. For example, in the loop sequence UUCG, the conformation of the backbone and the nucleobases allows for the formation of an additional Wobble base pair (G»U) within the loop and the stacking of the remaining loop nucleobases, thereby increasing the thermodynamic stability of the stem.

Rna Secondary Structural Motifs

Figure Box 10.1.2. Examples of secondary and tertiary structure motifs. The backbone is represented by the black line and the nucleobases by the light-gray short lines. Two light-gray lines facing each other represent a base pair. In the base triple (UAU) the A^U is a Watson- Crick base pair whereas U^A is not.

otides might have been delivered by comets and meteorites after having been synthesized under conditions (temperature, pressure, pH) completely different from those we infer for the primitive Earth. This proposition may well be relevant ifthe amount ofcarbon compounds delivered by extraterrestrial bodies (over 1016 kg during the several first 100 million years following the Earth formation) is considered. (ii) RNA could have been preceded by another more prebiotic molecule, component of a preRNA World that invented the RNA World. (iii) The RNA World might have emerged from a network of catalytic reactions using prebiotic organic molecules to produce more complex molecules. In this case, the system information was intrinsic to the interconnections of the catalytic processes. This idea is often referred to as the "Metabolic World."

The idea of a preRNA World (ii) has led to the investigation of several information polymers with different backbones, deemed simpler to synthesize (Fig. 10.3). It is assumed that this hypothetical RNA ancestor should efficiently form base pairs with itself and RNA monomers in order to obtain information transfer between the two Worlds.

Eschenmoser and his collaborators investigated in depth nucleotide derivatives based on variations of the sugar ring size11 to try to understand the reasons underlying the choice of ribose and deoxyribose in natural nucleic acids. Others designed new polymers that could solve some of the synthetic/structural issues of RNA: for example, nucleic acid derivatives with a peptide12 or a glycerol-phosphate13 backbone. Such derivatives are interesting for gaining insights into RNA properties. Although it is claimed that some of them, such as GNA and PNA, could be prebiotically plausible, no prebiotic syntheses, let alone processes for their non-enzymatic polymerization, have yet been reported. Moreover, some obstacles to the realization of these processes are already well documented, for example, the cyclization of PNA monomers into cyclic dimers.

In summary, there are no satisfactory de novo syntheses for P-D-nucleotides reported yet, which might reflect our poor understanding of prebiotic conditions or may well support the idea that the RNA World was not the first step in the evolution leading to cellular life. However, even if the "Metabolic World" postulate is correct, the catalytic network information would have to undergo transition at some point to an information/catalyst polymer-based form. Thus the study of non-enzymatic RNA polymerization and replication remains essential to an understanding of this process.

(ii) Reactivity of RNA Monomers

To ensure the polymerization of RNA monomers, it is crucial to understand their reactivity. The standard free energy for the synthesis of a phosphodiester bond is approximately +5.3 kcal/mol in aqueous solutions. Thus polymerization will not spontaneously occur unless the monomers are chemically activated (i.e., they are provided with chemical energy stored in the bond between the monomer and an activation group), or external energy (e.g., heat) is supplied. To activate RNA monomers, inorganic polyphosphates (Fig. 10.4) can be used, as they have been shown to promote the polymerization. However, because of their slow polymerization rates, most ofthe "prebiotic" polymerizations have been and still are, carried out using nucleotides activated as phosphoramidates, usually phosphorimidazoles (Fig. 10.4), even though these molecules might not have been present on early Earth.

Natural RNA is generally linked with 3'-5' linkages, a regioselec-tivity (Fig. 10.4) that is ensured by polymerase enzymes. Metal-ion mediated, non-enzymatic polymerization tends to yield a mixture of all possible linkages, thus producing RNA analogs. (When we speak of RNA in the remainder of this chapter, we will refer to the mixtures ofRNA and its analogs). Their relative frequency depends on the relative reactivity of the nucleophilic hydroxyl groups and also on the type of metal catalysts and the medium used. The heterogeneity of linkages in non-enzymatically synthesized oligomers could prevent RNA function and replication.

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