We are interested in the chemical and physical processes that facilitated the transition from chemical evolution to biological evolution on the early earth. As a way of exploring these processes, our laboratory is trying to build a synthetic cellular system that undergoes Darwinian evolution. Our view of what such a chemical system would look like centers on a model of a primitive cell, or protocell, that consists of two main components: a self-replicating genetic polymer and a self-replicating membrane boundary (Figure 1). The job of the genetic polymer is to carry information in a way that allows for both replication and variation, so that new sequences that encode useful functions can be inherited and can further evolve. The role of the protocell membrane is to keep these informational polymers localized, so that the functions they encode lead to an advantage in terms of their own replication or survival. Such a system should, given time and the right environment, begin to evolve in a Darwinian fashion, potentially leading to the spontaneous emergence of genomically encoded catalysts and structural molecules.
We hope that our explorations of the chemistry and physics behind the emergence of Darwinian evolution will lead to explanations for some of the universal properties of modern cells, as well as explanations of how modern cells arose from their simpler ancestors. As we explore these fundamental questions we are also on the lookout for chemical or physical phenomena that might have practical utility in biomedical research.
The first cells would have required a cell membrane that was very different from the membranes of modern cells, which rely on sophisticated protein channels and pumps to control the influx and efflux of small molecules. Since primitive cells lacked highly evolved protein machinery, their membranes would have had to allow nutrients to diffuse into the cell spontaneously. Similarly, membrane growth and division would have had to proceed in the absence of sophisticated biological machinery. How might this have been possible? Membrane vesicles built from simple amphiphilic molecules such as fatty acids are excellent models for the compartment boundaries of primitive cells because of their dynamic properties. The growth of such vesicles turns out to be relatively simple, and Pier Luigi Luisi's lab (then at the Swiss Federal Institute of Technology, Zurich) first showed that fatty acid vesicles can grow by incorporating additional fatty acid supplied in the form of micelles. By combining that process with a procedure for division by forcing large vesicles through small pores, we demonstrated multiple generations of vesicle growth and division, albeit in a rather artificial manner.
We have recently discovered a more prebiotically realistic process that leads to the coupled growth and division of larger vesicles. In this process, vesicles grow into long, fragile, thin filamentous structures. These readily divide into multiple smaller daughter vesicles in response to gentle agitation, such as might result from waves on a pond (Figure 2; Movie 1). To obtain a better understanding of the underlying processes that drive growth and division, we are continuing to study this pathway in more detail.
Concurrently with our work on vesicle replication, we are addressing the other major challenge in the synthesis of a protocell—the development of a self-replicating genetic polymer. Despite many years of effort, complete cycles of chemical (i.e., nonenzymatic) replication of RNA have not been achieved. It is possible, however, that small modifications of the structure of RNA might enable purely chemical replication. The late Leslie Orgel began to explore the polymerization of nucleotides with amino-modified sugars to form phosphoramidate nucleic acids in the 1970s and 1980s, but this work was not pursued, since the amino nucleotides did not appear to be prebiotically plausible. We have decided to explore a series of phosphoramidate-linked nucleic acids as an expedient means of obtaining a chemical replication system. For each polymer, we are synthesizing oligonucleotides for use as templates and for assessing their base-pairing properties in cases where these are currently unknown. In addition, we are synthesizing activated monomers for use in template-directed primer-extension experiments.
Our most promising system to date involves the polymerization of 5′-activated 2′-amino-dideoxyribonucleotides into 2′-5′-linked phosphoramidate DNA. These 2'-amino monomers are of particular interest because they cannot cyclize, and thus last much longer than many other monomers. We have found that the activated G monomer results in efficient nonenzymatic primer extension across oligo-dC templates, generating full-length copies in a matter of hours. We are examining the copying of a wide range of templates by these monomers and preparing to study self-replication in this system. By characterizing this and other synthetic genetic polymers, we hope to discover a complete, robust system for the replication of genomic sequences.
Our future work will be directed toward the synthesis of nucleic acids with more efficient and accurate chemical replication, and toward the discovery of more prebiotically plausible replicating genetic polymers. This chemical approach to genomic replication broadens the possibilities for the first genomically encoded catalysts, because they are not necessarily involved in replication but could play a role in other processes, such as metabolic activities.
The integration of replicating informational polymers with a replicating compartment boundary system provides numerous opportunities for both positive and negative interactions. Clearly the two subsystems must be compatible for the protocell as a whole to reproduce and evolve. One of the most important interactions involves membrane permeability, because the replication of a protocell genome can only occur if the corresponding nucleotide building blocks can get into the cell. We have studied the effects of membrane composition on permeability to small polar molecules such as the sugar ribose, and to larger, charged solutes such as nucleotides. We found that membrane components that increase disorder in the membrane, such as unsaturated or branched chain lipids, increase permeability. Lipids with larger head-groups, and thus an overall conical shape, also increase permeability, probably by stabilizing highly curved defects.
We found two membrane compositions that generate robust model protocell vesicles with high nucleotide permeability. The first is a convenient laboratory model consisting of myristoleic acid (C14:1) together with its glycerol ester. The second, which is more prebiotically reasonable, is composed of the short-chain, saturated fatty acid decanoic acid, its glycerol ester, and decanol. We used both types of vesicles to demonstrate the nonenzymatic copying of encapsulated templates following the addition of activated nucleotides to the external solution. The primer-extension reaction works well when the primer template is encapsulated in vesicles, with full-length product accumulating only slightly more slowly than in free solution, due to the added time required for the nucleotides to cross the membrane. In addition to being a major step toward the synthesis of a complete protocell, this experiment is significant because it implies that early protocells could have been heterotrophs that grew by taking up nutrients that were synthesized in the external environment.
The genetic material and the protocell membrane must both be compatible with a common set of conditions that drive the protocell through a complete cell cycle. Complete genomic replication requires a means of strand separation so that the duplex product of template copying is converted to single-stranded templates for the next round of copying. For templates replicating in solution, the simplest solution to this problem would be thermal strand separation, as in PCR (polymerase chain reaction). In a protocell model, however, thermal cycling would only be viable if the protocell membrane is not disrupted by heating. We therefore investigated the thermostability of membranes composed of mixtures of fatty acids with their glycerol esters and the corresponding alcohols.
To our surprise, vesicles made from some mixtures could be boiled for at least an hour without detectable loss of encapsulated DNA contents. We showed that encapsulated duplex DNA is in fact denatured by heating, suggesting that multiple rounds of template copying could be mediated inside vesicles by thermal cycling. Furthermore, membrane permeability to molecules such as nucleotides greatly increased at high temperatures. These experiments support the plausibility of a primitive, environmentally driven cell cycle, in which the chemical replication of internal genetic material, as well as vesicle growth and division, would proceed at low temperature. Brief high-temperature excursions, perhaps resulting from convective flow over geothermally heated rocks, would then mediate strand separation and nutrient influx, followed by further growth and strand copying.
Our current work is aimed at extending our protocell model by demonstrating complete cycles of template replication within replicating vesicles. If we are able to reach that goal, we hope to observe the spontaneous evolution of adaptive innovations in this relatively simple chemical system. The nature of such adaptations may provide clues as to how modern cells evolved from their earliest ancestors. Ultimately this research may also tell us whether the conserved biochemistry of life is driven by chemical necessity, or whether biochemically very different forms of life are also possible.