RNA Polymerization or Self Condensation of RNA Monomers

To qualify as "prebiotically plausible", any RNA polymerization process should likely occur

• in an aqueous environment or at least be supplied with its reacting species from an aqueous medium,

• in the presence of a complex mixture of monomers at low initial concentrations,

• and in the presence of metal-ion catalysts either as dissolved ions or on solid mineral surfaces.

• Furthermore, the polymerization rates must be higher than the decomposition rates of the polymeric products. This

Figure 10.3. Examples of alternative monomers (dark highlight). A) Glycerol nucleic acid, (S/R) GNA; B) Threose nucleic acid, TNA; C) Ribose or deoxyribose nucleic acid depending on the 2' group (OH versus H), RNA and DNA; D) pyranosyl RNA, pRNA and E) Peptide Nucleic Acid, PNA. (A) and (E) backbones are not sugar based. B, D and E can base-pair with themselves. A, B and E can base-pair with C, the natural nucleic acids.

Figure 10.3. Examples of alternative monomers (dark highlight). A) Glycerol nucleic acid, (S/R) GNA; B) Threose nucleic acid, TNA; C) Ribose or deoxyribose nucleic acid depending on the 2' group (OH versus H), RNA and DNA; D) pyranosyl RNA, pRNA and E) Peptide Nucleic Acid, PNA. (A) and (E) backbones are not sugar based. B, D and E can base-pair with themselves. A, B and E can base-pair with C, the natural nucleic acids.

Figure 10.4. Reactivity of activated p-D-ribonucleotides. A) Examples of activated monomers. B) The three products of RNA monomer dimerization have differing regioselectivity: (I) 5'-5' or pyrophosphate, (II) 2'-5' and (III) 3'-5'. N stands for any nucleobase.

Rubinetti Dwg

Figure 10.4. Reactivity of activated p-D-ribonucleotides. A) Examples of activated monomers. B) The three products of RNA monomer dimerization have differing regioselectivity: (I) 5'-5' or pyrophosphate, (II) 2'-5' and (III) 3'-5'. N stands for any nucleobase.

kinetic aspect is important as polymerization reactions tend to be inhibited in an aqueous environment due to entropic effects.

• Finally, the reaction process must selectively produce a pool of polymers with a high potential for activity.

We will now review the various approaches investigated to perform RNA polymerization from monomers, also called monomer self-condensation, which in part fulfill the requirements listed above.

(i) Self-Condensation in Homogeneous Aqueous Media

It was expected that activated nucleotides in the presence of dissolved divalent metal-ion catalysts (e.g., magnesium, lead or uranyl ions) would form extended stacks (which bring the reacting groups in close proximity) due to the relatively hydrophobic character of the heterocyclic nucleobases and the interactions of the divalent metal-ions with the negatively charged phosphate and ribose hy-droxyl groups (Fig. 10.5).

In principle, the activation groups with the metal-ions should permit one to overcome the energy barrier thereby allowing polymerization to occur. However, at low concentrations of catalyst and monomers, the products are primarily dimers with pyrophosphate and 2'-5' linkages. Even implementing a scenario such as the tidal pool (this scenario proposes concentrated mixtures of activated monomers and metal ion catalysts in small pools on beaches after the evaporation of the water), reactions only yield few products up to the 4-mers.14 This is in part due to the hydrolysis of the active species and to the incapacity of the hydrophilic reactive molecules (in particular, U residues) to form stacks. Oligomer elongation either by monomer addition or by ligation of short oligomers is

Figure 10.5. Schematic representation of RNA-monomer self-condensation. I) Self-assembly of the stacks. II) Phosphodiester bond formation catalyzed by metal-ion.

equally unsuccessful. That is, an oligomer length that would allow for product activity cannot be reached by non-enzymatic synthesis in homogeneous aqueous solution.

In the light of these unsuccessful attempts, it was surmised that the homogeneous aqueous set-ups do not allow for the ordering of the reacting species by stacking. Thus, a specific environment had to be introduced in the system, which would interact with the monomers and catalysts and concentrate them, thereby reducing water molecule activity and simultaneously ordering them. To date, three environments have been investigated: mineral surfaces, the eutectic phases in water-ice and lipid-bilayer lattices.

(ii) Self-Condensation on Mineral Surfaces, Such asMontmorillonites

The charged nature and regular structure of mineral surfaces can provide a supporting environment for polymerization of RNA. Various inorganic minerals, such as hydroxylapatite (Ca5(PO4)3OH) and clays such as montmorillonites (Fig. 10.6), have been used to promote RNA polymerization. Of the various generic clays tested, most showed little or no activity. However, dispersions of chemically treated montmorillonites, called ho-moionic, demonstrate significant performance.15

Activated nucleotides can adsorb either directly on the surface (via hydrophobic interactions or electrostatic interactions between their positively charged activating groups and the negatively charged surface) and/or be held there by metal-ion bridges between the nucleotide phosphates and the surface. The extent of the adsorption will be dependent on the properties of the activating group, the mineral surface and the aqueous solution. Furthermore, polymers adsorb more tightly than individual monomers due to their multiple cohesive interactions.

The montmorillonite-supported ImpA self-condensation in aqueous electrolyte solutions containing MgCl2 (the catalyst) produces RNAs of up to 50 monomeric units in length when small oligomers acting as primers (short deoxyribose oligomers with a last ribonucleotide, (pdA)9pA, or pyrophosphate ribonucleotides dim-ers, AppA) are present. In such experiments, activated monomers need to be regularly added (once a day for 14 days) to replenish the monomer stocks and prevent the growth-limiting effect of monomer hydrolysis. In contrast, when using monomers with large heterocycles as their activating group, such as 1-methyladenine, a comparable oligomer length is attained without primers within only 3 days. Moreover, all four nucleotides are incorporated.16

This polymerization is selective both at the level of the product nucleobase sequence and the regiochemistry of the linkages (see Box 10.2 for explanation concerning the experimental determination of these parameters). For example, while all possible dimers are formed in ImpA-ImpC mixtures, only a small number of all longer oligomer isomers predicted by the random synthesis are observed.17 The 3 ' terminal nucleobase (purine or pyrimidine) of the nascent oligomer and its regiospecifity determine the reactivity for elongation. Because a 3 ' -5 ' -linked terminal nucleotide elongates more efficiently, the ratio of 2 '-5 ' or 3 '-5 ' linkages observed on montmorillonite is greatly modified in favor of the natural isomers (3 ' -5 ' ) compared to that in a homogeneous aqueous medium where the 2 -5 linkage is predominant. Finally, since a 3 terminal purine has a higher intrinsic reactivity than that ofa 3 ' terminal pyrimidine, the formation of purine-rich oligomers is favored.

(iii) Eutectic Phase in Water-Ice

Exploration of planetary bodies in recent years has clearly established that water exists elsewhere in the Universe often as ice. On the primitive Earth, the solar irradiation was less intense (approximately 30% weaker) than today; thus water-ice mixtures might have been present.

Water-ice systems represent an interesting environment for RNA monomer self-condensation because such a system can very efficiently concentrate solutes (Fig. 10.7), thereby enabling their organization. Freezing reduces the water activity in the system by dehydrating the sample and owing to the low temperatures it should protect RNA products from thermal decomposition. The exact processes involved (simple concentration effects, interactions with the ice-surface, etc.) are not yet elucidated, but polymerization is only successful if ice nucleation occurs. Supercooled solutions are not conducive to efficient reaction.

When mixtures containing all four imidazole-activated monoribonucleotides in the presence of metal-ions, typically mixtures of Mg(II)/Pb(II), are incubated for up to 36 days at -18.4°C (a temperature slightly below the freezing point), quasi-equimolar, almost-complete incorporation (up to 90%) of all monomers

Figure 10.6. Montmorillonite structure and its influence on RNA polymerization (adapted from ref. 16). Montmorillonite is composed of two tetrahedral layers of silicates linked by octahedral aluminates. Si4+ and Al3+are partially substituted by alkali metal or alkaline earth metal (e.g., Mg(II)), as well as Fe(III) ions, due to weathering. The homoionic montmorillonite form is obtained when all exchangeable cations are replaced by Na+ions. Reactions take place on the surfaces and not on the edges.

Figure 10.6. Montmorillonite structure and its influence on RNA polymerization (adapted from ref. 16). Montmorillonite is composed of two tetrahedral layers of silicates linked by octahedral aluminates. Si4+ and Al3+are partially substituted by alkali metal or alkaline earth metal (e.g., Mg(II)), as well as Fe(III) ions, due to weathering. The homoionic montmorillonite form is obtained when all exchangeable cations are replaced by Na+ions. Reactions take place on the surfaces and not on the edges.

Box10.2. RNA Analytics

RNA strands produced by non-enzymatic polymerization can be analyzed in terms of length, nucleobase content and linkage regio-specificity using several methods.

Gel Electrophoresis. This method is used to determine the length of RNA products (greater than 5-mers) by comparison with a RNA ladder that contains RNA molecules ofknown length. A resolution of one nucleotide can be achieved. Also, the type oflinkage for a primer elongated by one nucleotide can be determined.

Chromatography. High pressure liquid chromatography (HPLC) using both reverse-phase and ion-exchange columns is used in RNA analytics. With reverse-phase chromatography, it is possible to separate dimeric products both according to their nucleobase content and their type of linkage (2'-5', 3'-5' and pyrophosphate). This type of analysis is performed to analyze the enzymatic decomposition products of RNA polymers and determine the ratio of natural to unnatural linkages. This methodology can also be applied to determine the nucleobase content of RNAs that have been completely decomposed into nucleotides under alkaline conditions or by phosphodiesterases. Ion-exchange chromatography is used to determine the length of short oligomers and their regiospecificity.

Enzymatic Digestion. Ribonuclease or RNase enzymes specifically cleave 3 -5 linkages and can be sequence-specific both in terms of nucleobase and the RNA structure (single strand or duplex) flanking the cleavage site. These enzymes can help us to determine the ratio of natural to unnatural linkages in conjunction with gel electrophoresis or chromatography.

Mass Spectrometry (MS). In particular, MALDI-TOF MS is used to determine the sequences of short oligomers. This method permits one to analyze the weight of biopolymers without destroying them. The resolution in mass can be as low as 1 proton mass, which allows for the relatively precise determination of sequence identity.

Sequencing. This methodology allows for the determination of nucleobase sequence in natural RNA and DNA. Nowadays, RNA fragments are enzymatically reverse-transcribed into cDNA or complementary DNA. This cDNA is then amplified, inserted in plasmids that are transfected in bacteria for further amplification. The DNA plasmid product is then sequenced. The mixture of linkages in RNA products synthesized non-enzymatically renders the reverse-transcription extremely difficult.

into medium-length mixed oligomers (up to 15- to 30-mers) is observed.18 Contrary to clays, the reaction occurs in the presence of various heterogeneous mixtures of activation groups and metal catalysts at initial concentrations of reacting species as low as 10-6 M. Usually, mixtures of Mg(II)/Pb(II) are used, but Pb(II) ions alone are catalysts for the polymerization at a ratio of catalyst to monomers as low as 1:20.19 Low concentrations of activated oligomers can effectively be ligated to yield longer polymers or elongated by adding fresh activated monomers. Interestingly, the elongation using monomer addition only proceeds beyond a range of length between 30- to 35-mers if the elongated oligomers are forming secondary structures (Monnard and Szostak, unpublished observations). This fact may indicate a form of selectivity toward structure-forming products, which could lead to a higher number of RNA catalytic species.

Product regiospecificity (3 '-5' linkage) is also improved compared to homogeneous aqueous reactions.18 Furthermore, the eutectic phase in water-ice has the advantage of reduced hydrolysis rates. In typical reactions, only 6-10% of an initial ImpU concentration is hydrolyzed to the inactive uridine monophosphate. In contrast, in homogeneous aqueous solutions and on montmorillonite hydrolysis is accelerated by metal ions in direct proportion to the monomer concentration by up to 600-fold.19 The low hydrolysis rate in the eutectic could have been essential for the generation and selection of long oligomers in an environment likely to have a limited monomer supply.

(iv) Lipid-Bilayer Lattices

The investigation of this particular microenvironment was spurred by the insight that chemical activation of the mononucle-otides may not be required if synthesis of phosphodiester bonds could be driven by the chemical potential of fluctuating anhydrous and hydrated conditions, with heat providing activation energy during dehydration. Amphiphile bilayers that compose vesicles or liposomes could represent a model for such a fluctuating environment. Under the right conditions, bilayer structures are formed

Figure 10.7. Formation of the eutectic phase in water-ice. A dilute solution of activated monomers (ImpN) and metal-ion catalysts (M2+) is frozen below its freezing point, but above the eutectic point (i.e., the temperature at which the whole sample is frozen-solid). During freezing, solutes are concentrated in the liquid phase (eutectic phase) between the pure-ice crystals (see the light micrograph of a reaction mixture containing a fluorescent dye). Note that upon the initiation of freezing, the concentration of the solutes increases which simultaneously lowers the freezing point of the residual solution. Bar = 26.7 |m.

Figure 10.7. Formation of the eutectic phase in water-ice. A dilute solution of activated monomers (ImpN) and metal-ion catalysts (M2+) is frozen below its freezing point, but above the eutectic point (i.e., the temperature at which the whole sample is frozen-solid). During freezing, solutes are concentrated in the liquid phase (eutectic phase) between the pure-ice crystals (see the light micrograph of a reaction mixture containing a fluorescent dye). Note that upon the initiation of freezing, the concentration of the solutes increases which simultaneously lowers the freezing point of the residual solution. Bar = 26.7 |m.

Figure 10.8. Formation of the lipid-bilayer lattices. RNA monomers (NMP, ribonucleotide 5'-monophosphate) are mixed with a liposome suspension (lipid bilayers are the circular and ladder-like columnar structures). Upon dehydration of the mixture, liposomes fuse into multi-layered structures forming a lipid-bilayer lattice that retains some moisture while sequestering monomers in extended stacks (gray line).

Figure 10.8. Formation of the lipid-bilayer lattices. RNA monomers (NMP, ribonucleotide 5'-monophosphate) are mixed with a liposome suspension (lipid bilayers are the circular and ladder-like columnar structures). Upon dehydration of the mixture, liposomes fuse into multi-layered structures forming a lipid-bilayer lattice that retains some moisture while sequestering monomers in extended stacks (gray line).

when amphiphile molecules are suspended in an aqueous medium. This molecular arrangement is preserved when vesicles are dehydrated. If small molecules, such as RNA monomers, are present in the aqueous medium during the dehydration, they will be trapped between the amphiphile bilayers (Fig. 10.8) and likely ordered into a structure potentially conducive to polymerization.

There are several main differences between this approach20 and the two previously described ones: (A) The monomers are not chemically activated. (B) No metal-ion is used as a catalyst. As a corollary, the RNA linkage formation must be induced by an external energy source, e.g., heat. (C) The use of amphiphile bilayer lattices ensures that the system remains in a fluid state. (D) A fraction of the RNA products will become compartmentalized at the end of the procedure. We will come back to this latter point.

In experiments conducted by Deamer and collaborators,20 phosphatidylcholine lipids (these molecules seem unlikely on the prebiotic Earth) dissolved in ethanol were injected into an aqueous solution containing nucleotide monophosphate where they spontaneously formed membranous structures. The suspension was then heated, dried under a flow of carbon dioxide forming lipid-bilayer films and incubated for up to 2 h at temperatures between 70 and 90°C. The samples were then rehydrated and the drying-heating-rehydrating cycles were repeated up to 7 times.

With samples containing only one nucleotide monomer, adenine 5'-monophosphate (AMP) or uridine 5'-monophosphate (UMP), RNA molecules with lengths between 50-100 monomeric units are detected that represent maximally 10% yield ofpolymers by weight. The maximum yields depend on several experimental parameters: temperature, cycle number, the type ofgas used during dehydration of the suspension, the lipid composition and the lipid to nucleotide molar ratio. Mixtures of UMP and AMP produce polymers of reduced length, which match those observed on montmorillonite or in the eutectic phase in water-ice. In the absence of lipids but with the same cycling, no products longer than tetramers can be detected. Finally, the regioselectivity of the RNA products remains unclear as the researchers only stated "products could not be digested by RNase enzymes."20

In summary, the results obtained with these three different approaches to the polymerization of RNA from monomers clearly indicate that multiple pathways can exist to yield polymeric molecules with a length compatible with an RNA activity. These polymers still contain mixed linkages, a fact that may well inhibit their catalytic activity and their replication.

0 0

Post a comment