Non-enzymatic nucleic acid polymerization and replication

Primary investigator: Pierre-Alain Monnard.

Biopolymers such as nucleic acids and proteins, play an essential role in cellular life, as information, catalytic and structural molecules. Analogous polymers must have played a similar role in the emergence of Life on the early Earth or other planetary bodies and some scenarios proposed to explain the origins of Life, most prominently among them the RNA World, envision polymers as the core molecules in the events leading to Life.

The RNA World hypothesis was first advanced in the 1960s by F.H.C. Crick and L.E. Orgel among others and later named by W. Gilbert. These authors postulated an autonomous RNA-based "organism" or protocell if RNAs could perform several catalytic functions presently carried out by proteins, first and foremost that of RNA replication, while acting as genetic information repository. Although the RNA World seems ineluctable at some point during the emergence of cellular life from inanimate matter, it is not clear whether it represented the first system during Evolution towards Life.

The realization of a functional RNA World requires a series of events including A) the abiotic synthesis of RNA monomers and their activation and B) their assembly into oligomers (in the likely presence of metal catalysts) that could be further elongated C) by ligation of additional monomers or another oligomer. The catalytic activity expected from the RNA polymers requires relatively long polymers (at least 20-30 residues) and often 100 or more nucleobase fragments are needed during selection for complex activities. D) These molecules would have had to serve as templates for their own spontaneous copying or replication. E) At that stage, a pool of RNAs (a large set of RNA molecules) must have existed from which a set of catalytic RNAs (among them, RNA-based RNA copying molecules) emerged through natural selection that together sustained their exponential growth in the prebiotic environment.

Our interest lays in understanding how

Scheme 1: Schematic representation of RNA-monomer self-condensation. I) Self-assembly of the stacks. II) Phosphodiester bond formation catalyzed by metal-ion.

i) RNAs can be formed from their monomers by condensation, which is not necessarily straightforward in an aqueous environment but can occur in a micro- or nano-structured medium (form of heterogeneous catalysis)
The main question here is to understand how the polymerization process can occur and deliver RNA products of length compatible with RNA activity. Once a reliable methodology is established whether the process delivers us a relatively heterogeneous set of RNAs (in terms of the sequences) and whether RNAs with a specific activity, such as ligases, can be detected and isolated from this RNA fragment set.

In homogeneous aqueous solutions under "prebiotically plausible" conditions, it is rarely possible to convert a significant proportion of the input of activated monomers to oligomers that are 10 monomer units long or longer, even when starting from highly concentrated solutions. In contrast, biopolymer synthesis at low monomer concentrations proceeds quite efficiently within cellular aqueous volume by relying on compartmentalization and protein enzymes. Whereas enzymes were not a viable option on the early Earth, the "compartmentalization" of the RNA polymerization could also be achieved by chemical and physical means by using for example clays or set-ups obtained when conditions (temperature, or pressure, or solution composition) of a chemical system are changed so as to promote the formation of multiple intertwined phases in the medium. For example, cooling an aqueous solution below their freezing point, but above their eutectic point (the temperature at which the solution is frozen solid (Scheme 3A)1 or using the self-assembly of lipids in solution (Scheme 3B) and drying the resulting structures will yield such a compartmentalization system.

Scheme 3. A) Lipid-bilayer lattice supported polymerization. A solution containing activated monomers (ImpN, Imidazolide-activated ribonucleotide) and metal catalysts is mixed with a liposome suspension (lipid bilayers are the light-gray lines). Upon dehydration of the mixture, liposomes fuse into multilayered structures forming a lipid-bilayer lattice that retains some moisture (this is not the case in the absence of lipid) while sequestering monomers and metal-ion catalysts in extended stacks (dark-gray lines). B) Water-ice supported polymerization. A dilute solution of activated monomers (ImpN) and metal-ion catalysts is frozen. During freezing solutes are concentrated in the liquid phase (eutectic phase) between the pure-water crystals (see the light micrograph of reaction mixture containing a fluorescent dye).

In these "compartmentalization" systems, the objectives are to concentrate the activated RNA monomers and metal-ion catalysts out of the bulk aqueous medium and to permit the formation of monomer assemblies, such as stacks that are conducive to polymerization. Furthermore, these environments can preserve both the activated state of the monomers prior to reaction and prevent the degradation of the RNA polymers by hydrolysis. A non-enzymatic RNA polymerization could also permit the synthesis of chemically modified RNAs that could well extend the activity spectrum over their natural counterparts have a limited usage due to polymerase substrate specificity.

Kanavarioti, A., Monnard, P.-A. and Deamer, D.W.: (2001) Eutectic phases in ice facilitate the oligomerization of phosphoimidazolide activated mononucleotides. Astrobiology, 1, 271-281.
Monnard, P.-A., Apel, C. L., Kanavarioti, A. and Deamer, D. W.: (2002) Influence of ionic solutes on self-assembly and polymerization processes related to early forms of life: Implications for a prebiotic aqueous medium. Astrobiology, 2, 139-152.
Monnard, P.-A., Kanavarioti, A. and Deamer, D.W.: (2003) Eutectic phase polymerization of activated ribonucleotide mixtures yields quasi-equimolar incorporation of purine and pyrimidine nucleobases. J. Am. Chem. Soc., 125, 13734-13740.
Monnard, P.-A.: (2005) "Catalysis in abiotic structured media: An approach to selective synthesis of biopolymers."Mol. Cell. Life Sci., 62, 520-534.
Monnard, P.-A. and Szostak, J. W.: (2008) Metal-ion catalyzed polymerization in the eutectic phase in water-ice: A possible approach to template-directed RNA polymerization. J. Inorg. Biochem., In press.
Monnard P.-A. and Ziock H.-J.: Eutectic phase in water-ice: A self-assembled environment conducive to metal-catalyzed non-enzymatic RNA polymerization. Chemistry and Biodiversity, Accepted.
Monnard, P.-A.: Elongation of RNA oligomers in eutectic phase in water ice: Towards the creation of long-RNA pools. In preparation

Spatial exploration undertaken by NASA has also established the ubiquitous presence of water in the form of ice in the universe as, for example, the ice deposits on Mars, the ice crust covering a liquid ocean for Europa and Enceladus, and as a major component of comets. Essential simple chemicals for the emergence of Life have already been synthesized in ices that are thought to accurately model interstellar ices.