Lipid Encapsulation of Self-replicating Ribozymes

Andrew Jones

December 2012 Department of Biology Carthage College

A Senior Thesis for the Partial Fulfillment of the Degree of BACHELOR OF ARTS in Biology

by

Andrew Jones Class of 2013

DEPARTMENT OF BIOLOGY Carthage College Kenosha, Wisconsin

__________________________________________________ Dr. Deanna Byrnes, Senior Seminar Instructor Department of Biology

__________________________________________________ Dr. Patrick Pfaffle, Thesis Advisor Department of Biology

Abstract A self-sustaining protocell with an RNA based heredity system would be an important intermediate between prebiotic chemistry and what we would consider life. I aim to combine research done with RNA polymerase ribozymes and liposomes under plausible early Earth conditions. I would hope to observe sustained replication of both the ribozyme and the protocell. It would represent a necessary step forward for the RNA world hypothesis. Introduction The Last Universal Common Ancestor (LUCA) of all life on Earth has been described as a bacterial and archeal predecessor with an RNA genome (Glansdorff et al., 2008). However, a fullfledged protein translation system must have evolved in a step-wise manner, so something simpler must have come before. Beginning in the late 1960's with the elucidation of the DNA and protein translation system, the question of what came first was asked (Orgel, 2004). The interdependency of these systems created a chicken or the egg scenario that spurred further inquiry. Scientists like Carl Woese, Francis Crick, and Leslie Orgel realized that a replication system based only on nucleic acids was plausible due to base-pairing and the fact that no such system was possible using only proteins (Orgel, 2004). Shortly afterward, it was proposed that nucleic acid-containing coenzymes, like the ribosome, were living fossils of the RNA world. The RNA world hypothesis states that life began with RNA instead of DNA and that both genomic and enzymatic roles were fulfilled by RNA to start. Although it is much simpler than the DNA/protein system, there were still questions to be answered. Orgel outlines four problems that I will discuss relating to the origin of life: synthesis of nucleotides without relying on an enzyme, nucleotide polymerization without an enzyme, replication of RNA without an enzyme, and the emergence of a functional RNA catalyst (also referred to as an RNA enzyme or ribozyme) that would be subject to evolution and natural selection. I have also considered that a plausible lipid membrane is required. This would both protect the RNA from degradation and grant a means of heredity and lineage to the ribozyme inside. I propose that a lipid encapsulated, self-replicating RNA enzyme could have

occurred on the early Earth, given the presence of necessary organic molecules and plausible preenzyme polymerization and encapsulation mechanisms. Showing that such a system could have formed and could be subject to mutation and natural selection would contribute further knowledge and understanding to the fields of abiogenesis and evolution. The ribosome is a ribozyme, also known as an RNA enzyme, that is composed of either three or four RNA subunits ranging from 120-4500 nucleotides in length. The number of subunits depends on its evolutionary origin; prokaryotic ribosomes having three subunits and eukaryotic ribosomes having four subunits. The RNA subunits are also associated with 50-80 small proteins. Interestingly though, the RNA portion has been shown to be catalytically active on its own. The active site is an evolutionarily conserved adenosine within the RNA portion that forms peptide bonds through acid-base catalysis (Zhang & Cech, 1997; Barta at al., 2001), meaning that particular adenosine within the RNA portion has been important enough to the ribosome's function that it has been maintained throughout evolutionary history. The protein subunits are nonessential evolutionary additives. They serve as translation and feedback regulators, preventing translation termination by complexing with RNA polymerase in prokaryotes (so that the ribosome follows closely behind the RNA polymerase because prokaryotes have a simultaneous transcription/translation system). They also increase overall stability of the ribosome, making tRNA removal more efficient, which increases the rate of translation (Stelzl et al., 2001). The fact that the protein subunits only serve secondary functions and that the RNA portion can catalyze peptide bond formation on its own seems to indicate that the RNA portion evolved first, independent of the protein subunits, which further supports the RNA world hypothesis. Other ribozymes besides the ribosome have been discovered. Most do nothing more than cleave phosphodiester bonds (Pley et al., 1994), but some are capable of different functions. Rnase-P is an enzyme found in all organisms that converts precursor tRNA into active tRNA. Rnase-P is an RNA/protein complex like the ribosome and, like the ribosome, the RNA portion is active on its own (Li & Deutscher, 2002). Many studies lately have involved self-replicating ribozymes, of which there

are a couple variations. One is a single ribozyme that ligates two oligonucleotides together, resulting in a catalytically active ribozyme similar to itself (Joyce, 2009) and the other is a cross-catalytic system of two ribozymes- A and B. This means A catalyzes the formation of B and B catalyzes the formation of A (Lincoln & Joyce, 2009; Joyce, 2009). A key characteristic of these ribozymes is that their replication is not perfect. Since not all of the bases need to be base-paired for ligation, they possess a level of sequence generality and the potential for different mutant oligonucleotides joining together is entirely possible, creating a Darwinistic evolution and natural selection scenario. It is also the simplest selfsufficient system capable of doing so, because these ribozymes do not rely on proteins or any other template materials besides themselves. Recent experiments have created complex ecosystems performed both in vitro and in silico (Bentin, 2011; Joyce 2009; Takeuchi & Hogeweg, 2008). These experiments have examined the interactions and competition of different ribozyme phenotypes. Selection is a passive process in these systems; it is merely the result of ribozymes with higher fidelity and faster replicative speed being able to accurately base pair and quickly ligate their precursor oligonucleotides. Although they serve as functional models, in the context of early life and the beginnings of the RNA world, they present a glaring issue. Despite the ability of the non-base-pairing regions to have diversity, the base-pairing regions are specific to the ribozyme. An example of these cross-catalytic, oligonucleotide-ligating ribozymes is shown below in Figure 1. The inter-strand basepairing seen between E' and A and E' and B is necessary for proper positioning of the substrates so that ligation can occur. Without those specific sequences, base-pairing would be negatively affected, which would negatively affect replicative speed. Although they support some level of sequence generality in their oligonucleotide precursors, they still have relatively strict length and sequence requirements for binding and ligation, which is why they are considered models instead of simulations. This sequence specificity makes them extremely unlikely to occur naturally, as mineral-catalyzed ligation, like that performed by montmorillonite clay, would create oligonucleotides with random sequences (Ertem, 2004). For these reasons, I would not consider them as candidates for a likely early Earth scenario.

Figure 1: One of Joyce and Lincoln's ribozymes, E', base-paired to its complementary substrate oligonucleotides, A and B. The arrow indicates the ligation between A and B, which would create a functional E ribozyme. E would then ligate A' and B' to make another E' (Lincoln & Joyce, 2009).

I have found what I believe to be a more likely candidate for an early RNA world scenario: ligasederived RNA polymerase ribozymes that have been created by Lawrence & Bartel. Although Lawrence and Bartel had problems with fidelity, strides have been made with further evolution of these ribozymes just as more successful ribozymes had been made with the aforementioned oligonucleotideligating ribozymes (Zaher & Unrau, 2007; Bentin, 2011). The evolved Round-18 polymerase, B6.61 (shown below in Figure 2), is a ribozyme with a length of 189 nucleotides. Through in vitro selection, it has evolved to ligate >20 nucleotides of a template sequence. I believe through further in vitro evolution, this ribozyme could be capable of fully replicating itself by binding its own template sequence, which, when transcribed, would produce a working copy of the original without a reliance on oligonucleotides.

Figure 2: The secondary structure of the Round-18 polymerase, the polymerase which underwent amplification and evolution which resulted in the evolved B6.61 ribozyme. The conserved ligation site is shown in black, the ligase core in red, and the accessory domain in blue (Zaher & Unrau, 2007).

The lack of likely precursors, in the form of the specific oligonucleotides, is the distinguishing factor between plausible early Earth scenarios and the synthetic biology that Joyce and Bentin's work consists of. Since the days of Urey and Miller's famous experiment in which they synthesized amino acids by producing an electrical discharge in a reducing atmosphere, prebiotic synthesis routes for a variety of organic, prebiotic molecules have been studied. Plausible synthesis routes and confirmed extraterrestrial origins exist for all the necessary molecules, including pyrimidines, purines, simple sugars, sugar phosphates, and lipids (Callahan et al., 2011; Unrau & Bartel, 1998). A synthetic pathway has been described that bypasses the free ribose and nucleobase step to directly make activated pyrimidine nucleotides out of available cyanamide, cyanoacetylene, glycolaldehyde, glyceraldehyde, and inorganic phosphate. The inorganic phosphate is used as a general base catalyst while also being used in the reaction itself (Powner et al., 2009). Purine synthesis has been studied much more extensively than pyrimidine synthesis and has been outlined as the polymerization of hydrogen cyanide

at moderate temperatures combined with dehydration (Basile et al., 1984). A wide variety of nucleobases and other organic molecules (as well as hydrogen cyanide and ammonia) have been found in meteorites, specifically carbonaceous chondrites like the Murchison meteorite that fell in Murchison, Victoria in Australia (Callahan et al., 2011). The deposition of these molecules as meteorites impacted and fragmented would contribute to their presence in the seas so the aforementioned synthesis routes do not have to account for all of the molecules present on Earth. In addition to nucleobases, carbonaceous meteorites also contain amphiphilic molecules, or lipids. Exposure of interstellar molecular clouds and ice consisting of H2O, CO, CO2, CH3OH, NH3, and other compounds to high energy, ionizing radiation has been shown to form hundreds of complex organic molecules, like amino acids and amphiphilic molecules (Deamer et al., 2002). Although significant amounts of meteorites do not hit Earth anymore, around 4 billion years ago, there was a period of heavy bombardment which would have delivered large amounts of organic molecules to the surface. The importance of these lipids lie in their ability to spontaneously form micelles and, more importantly, liposomes. Amphilic molecules like lipids are characterized by the presence of a non-polar, hydrophobic hydrocarbon tail and a polar, hydrophilic head group. When in water, lipids spontaneously self-arrange to maximize hydrophobic interactions between the hydrocarbon tails and hydrophilic interactions between the heads and the water. A bilayer membrane is the lowest energy arrangement precisely for this reason. Liposomes have an innate tendency to split into daughter liposomes when they expand. Heterogenous internal and external pressures from replicating molecules in the interior and fluid pressure from the outside can work in conjunction to deform the liposome, meaning that the pressure on the membrane is not the same at every point around it, so it will have a tendency to deform and destabilize. As it extends and stretches in one direction , the middle will shrink and constrict, eventually splitting apart, leaving two separate daughter liposomes with the contents of the original liposome split between them (Stano & Luisi, 2010; Macia & Sole, 2007). Experiments have been done with liposomes and encapsulated biological molecules. One was done with PCR-amplified DNA,

which provided the internal pressure to induce self-reproduction of the liposome (Kurihara et al., 2011), and another used a short RNA genome that encoded for a replicase enzyme. In addition, the liposome contained all the necessary materials for translation of the replicase, so that a continuous cycle of replication and translation would occur (Kita et al., 2008). By combining the concepts of self-replicating liposomes with the evolved B6.61 ribozyme, one can imagine a self-sufficient protocell, which will expand with replication of the ribozyme and split into daughter liposomes once it reaches an unstable size (Kurihara et al., 2011). This would provide a system of heredity, as the offspring would have roughly half of the contents and faster replicating mutants would come to dominate the population. Compartmentalization also overcomes the dilute conditions of the large body of water by concentrating solute molecules and protects the RNA from external conditions which may degrade it (Deamer et al., 2002). Although the liposome would keep substrate molecules nearby by containing them, gathering them in the first place is also an obstacle. Fortunately, a number of plausible ways exist to concentrate prebiotic molecules. Wave and tidal action would bring them near the shore and deposit them on and near rocks. The evaporating lagoon hypothesis states that if lagoons or puddles are formed, evaporation of the water would concentrate the solute. Mineral surface adsorption might also play a factor. Adsorption is the association of a molecule with a surface through intermolecular forces. Without an active transport system for substrate molecules, a membrane made of long-tailed lipids like those used in Kita and Kurihara's studies would not allow precursor molecules to enter the liposome, starving the ribozyme. Luckily, the Murchison meteorite amphiphilic molecules (5-methylnonanoic acid) have a very short (eight carbons) hydrocarbon tail. In addition, the tail is branched, which would make the membrane slightly more permeable because the stacking capability of a branched chain is less than that of a straight chain. This leaky membrane would easily let in ionic solutes like activated nucleobases as they are used up by the ribozyme in an effort to achieve equilibrium. Considering that reducing the chain length of a bilayer from 18 carbons to 14 increases the permeability to ionic molecules by a factor of 1000 (Deamer et al.,

2002), an eight carbon lipid membrane would allow much more in than that while also sufficiently retaining larger macromolecules like the ribozyme itself. The size of the ribozyme presents a problem because a lipid bilayer would be impermeable to it. Since it can't diffuse through the membrane, an alternate method of encapsulating it is required. Both hydration-dehydration and freeze-thaw cycles have been shown to encapsulate large macromolecules. Due to the warm conditions of the early Earth as a result of the high CO2 concentration (Trainer et al., 2006), the former seems more likely. Interestingly, Kurihara's encapsulation method is a hydrationdehydration method, which makes the hydration-dehydration hypothesis more plausible. The early Earth's atmosphere lacked O2 (Trainer et al., 2006). Instead, the atmosphere consisted mainly of N2, CO2, CH4, H2O, and small amounts of H2. Another aspect of the early Earth is the presence of soluble iron, Fe2+, in the oceans instead of Mg2+, which was due to the reducing atmosphere. Magnesium is a common enzyme cofactor that plays an important role in the folding and catalytic properties of modern RNAs. Experiments replacing magnesium with iron as a cofactor have resulted in up to a 25-fold increase in catalytic activity (Athavale, et al., 2012). This lends more support to the RNA world hypothesis, because it would make ribozymes better by increasing their turnover number, which is the number of substrate molecules that an enzyme can convert per unit of time. To summarize, I hypothesize that the B6.61 can be evolved in vitro to be even better at extending and ligating a given template sequence, eventually to a point where it could replicate itself given a template sequence of itself, so that a self-sustaining catalytic system subject to mutation and natural selection would be produced. Granted that it would be possible to evolve B6.61, I would also like to encapsulate it to create distinct, self-replicating protocells. Another goal is to simulate an early Earth environment based on the evaporating lagoon hypothesis, which is a hypothesized method of prebiotic molecule concentration due to the gradual evaporation of water, to see if a ribozyme could be encapsulated by plausible natural processes and could rely on evaporation to overcome dilute conditions.

In light of the prior research demonstrating that the necessary prebiotic reactions can occur under hypothesized early Earth conditions, the emergence of a self-sustaining, encapsulated ribozyme system is both possible and would comply with the current, most widely accepted abiogenesis hypothesis- the RNA world hypothesis; it would represent an important stepping stone between prebiotic chemistry and what many believe to be the first life form. Between the synthesis routes outlined and the discovery of an RNA polymerase ribozyme, the literature satisfies Orgel's four problems. Methods In vitro evolution The B6.61 polymerase ribozyme will be evolved and selected according to Zaher and Unrau's methods (Zaher & Unrau, 2007). A library of approximately 1015 mutagenized ribozymes will be emulsified into water-in-oil droplets in sixty 50 mL conical tubes. RNase free water will be used to prevent degradation of the RNA. A 2.5 mL mixture of selection buffer consisting of 10 nM primertemplate, 40 mM Tris-HCl buffer, 2.5 mM spermidine (cofactor for T7 polymerase), 50 mM MgCl2, 0.01% Triton X-100 (a surfactant), 10 mM DTT, 8 mM GTP, 2 mM ATP, 2 mM CTP, 2 mM of 35Stagged UTP, and 10 units/L of the T7 polymerase will be emulsified in a 47.5 mL mixture of 0.5% Tween 80 and 4.5% Span 80 (also surfactants) in liquid paraffin (heavy mineral oil). The temperature will be maintained at 4C to inhibit any reactions from occurring before emulsification. The water and oil mixture will be emulsified by adding 6 mm glass beads and vortex-mixing for 5 minutes. The DNA templates added before emulsification will be created using mutagenic PCR, giving the population variety. These DNA templates will be transcribed after emulsification by the T7 polymerase for 3 hours at 37C. The polymerase will then be deactivated by heating the emulsion to 65C for 15 minutes. The emulsion will be allowed to incubate at room temperature (25C) for 18 hours before extraction and artificial selection of the most successful ribozymes. Extraction will be performed by centrifugation of the emulsion at 12,000g for 20 minutes. The

supernatant will be removed and saved. The pellet will be washed with 30 mM EDTA, resuspended by vortexing, and centrifuged again. The aqueous phase supernatant will be added to the supernatant from the original extraction. The supernatant will then be washed with diethyl ether and the ether will be discarded in order to remove any remaining oils. The nucleic acids in the aqueous phase will be extracted using a phenol/chloroform extraction and precipitated using ethanol. A phenol/chloroform extraction creates a bilayer with the phenol/chloroform layer on the bottom and the aqueous layer on top. The nucleic acids will remain suspended in the aqueous layer, while any remaining oils or other unwanted non-polar molecules will be dissolved into the phenol/chloroform layer. The aqueous layer will then be removed and put into another container. For each 10 mL of aqueous solution, 1 mL of 3 M sodium acetate and 25 mL of at least 95% ethanol will be added. The solution will be incubated on ice for 30 minutes. The solution will then be centrifuged at 12,000g for 20 minutes. The supernatant will be removed and the pellet, containing the ribozymes and products, will then be resuspended in water. Selection begins with denaturing of the resuspended nucleic acid by the addition of KOH to a final concentration of 50 mM. A biotinylated DNA probe complementary to the primer-template sequence will then be added. The solution will then be neutralized with Tris-HCl and streptavidin beads will be added. The beads will be washed with 0.5X SSC buffer. This buffer consists of 0.075 M NaCl and 7.5 mM trisodium citrate adjusted to neutral pH with HCl. The hybridized nucleic acid will be eluted from the beads by adding 50 mM KOH to them. The resulting solution will be neutralized with Tris-HCl and the nucleic acid will be precipitated with ethanol. The DNA will then be amplified using PCR. An APM (N-acryloyl-aminophenyl-mercuric acid) gel shift assay will be used to identify the longest primer extensions. APM gel slows the mobility of 4SUTP-containing nucleic acids so that those with longer primer extensions will appear higher on the gel. Autoradiography will be used to visualize the presence of 4SUTP in the gel, which will show where and to what extent the extended primers migrated.

Compartmentalization of the working ribozyme Once a ribozyme capable of fully replicating itself is produced, the hydration-dehydration method modified from Kurihara (Kurihara et al., 2011) will be used to create lipid encapsulated protocells containing the ribozyme. A solution will be prepared, including the ribozyme, 2 mM of each rNTP (ribonucleotide triphosphate), 40 mM Tris-HCl, 1 mM EDTA, and 50 mM MgCl2 (alternatively, FeCl2). This will be kept on ice to inhibit replication until compartmentalization. To test liposome formation with the meteoric lipids, a 20 mM 5-methylnonanoic acid solution will be prepared in CHCl3 in a test tube. The solvent will be evaporated under a nitrogen stream, leaving a thin lipid film on the inside of the test tube. The ribozyme-containing solution will be added to this test tube. The tube will be capped and vortex-mixed. This agitation will create liposomes containing the ribozyme solution. To ensure that replication only occurs inside the liposomes, RNase will be added to the solution to degrade any free-floating ribozyme. To accelerate replication, the tube will be allowed to warm to room temperature (25C). To confirm liposome formation, fluorescence and differential interference microscopy will be used. Figure 4 shows liposomes viewed with both methods. Negative controls will be done by creating liposomes without any ribozyme in the precursor solution to show that the replication of the ribozyme is necessary for liposome replication (Kurihara et al., 2011). Simulated evaporating lagoon and shoreline In addition to the test tube experiments, I propose to create a natural shoreline mimicking the early Earth. To comply with early Earth conditions, the container would have an airtight seal, so the atmosphere inside can be slightly reducing, in accordance to what the atmosphere was hypothesized to have been like 4 billion years ago (Trainer et al., 2006). The gas mixture inside would be mainly nitrogen gas with carbon dioxide and methane at roughly 1000 ppmv (parts per million by volume) each. Trace amounts (less than 100 ppm) of water vapor and hydrogen gas would also be present. The

shore itself would be constructed of granite and zircon rocks, along with montmorillonite clays (Hazen et al., 2008; Ertem, 2004). The water will be buffered by 40 mM Tris-HCl as with the other experiments. Due to the presence of reduced iron in the early Earth's oceans, FeCl2 will be added to the water at a concentration of 50 mM instead of MgCl2. A pump system will be used to simulate both rain and tidal action, cycling between wetting the rocks and drying them. To simulate a drying lagoon, the top will be made at a slant leading to a separate reservoir. This would enable the water to evaporate and condense without falling directly back into the lagoon. This evaporation would concentrate solute molecules as outlined by the evaporating lagoon hypothesis. Ten hydration-dehydration cycles should be sufficient to concentrate solute molecules and form liposomes. Anticipated Results The replicative ability of the evolved ribozyme will be analyzed with an APM gel. Longer transcribed sequences would appear higher on the gel relative to shorter sequences, meaning more effective ribozyme genes (those capable of successfully transcribing a longer template sequence) would be higher. Figure 3 shows a radiograph of the APM gel where black lines indicate the presence of the nucleic acids containing the 35S-tagged uracil. The evolved B6.61 ribozyme has a higher concentration of products with lengths in the 10-25 nucleotide range than the wild type ribozyme. This shows the increased replicative success of B6.61. Longer extension is indicated by higher bars on the gel. The control for this would be the unextended primer on its own as a baseline on the gel. This can be seen as the large black line at the bottom of the gel in Figure 3. Because I have no expected concentrations, the mere presence of higher bands indicative of longer primer extension is sufficient in indicating relative replicative success. Other possible results exist for the three experiments: the in vitro evolution could fail to evolve a better B6.61 ribozyme or it could stall at a particular number of nucleotides. The hydrationdehydration method may fail to produce viable liposomes or it may fail to encapsulate the ribozyme. The evaporative and tidal action of the simulated shoreline may also fail to produce any liposomes.

Figure 3: This APM gel shows the visualization of radiolabeled transcription products. The extent of transcription in nucleotides is shown on the right, with the longest primer extension being somewhere around 24 nucleotides in length (Zaher & Unrau, 2007). Liposome formation can be confirmed or denied by viewing a sample of solution placed on a

slide with a cover slip. The liposomes pictured below in Figure 4 are shown next to a scale bar which indicates 10 m. A structure with a radius of approximately 15 m is easily visible with a light microscope, seeing as an E. coli cell is about 2 m long and 0.5 m long and can be viewed relatively easily at 400x magnification. Liposome replication may even be seen under the microscope, as they would not require fixing to view. A replication rate fast enough to be seen under the microscope would confirm the encapsulation of the ribozyme, because the replication would be the result of the catalytic activity of the ribozyme causing pressure to build up inside the liposome. This method would be used to confirm liposome formation from both the in vitro test and the lagoon/shoreline. Due to the nature of such observational studies, I do not believe statistical tests are required.

Figure 4: These images show the progression of lipid vesicle replication. The top sequence shows the vesicles being viewed with differential interference microscopy while the bottom shows what they look like using fluorescence microscopy (Zaher & Unrau, 2007).

Discussion A number of obstacles still remain in regard to ribozymes as a spontaneously arising, selfsustaining replicative system. First and foremost is the current lack of a working RNA polymerase ribozyme. Given the relatively short amount of time that research like this has been done, however, the ligation of ~20 nucleotides by Zaher & Unrau and Bentin, or roughly 9% of the length of the full 187 nt (nucleotide) ribozyme, is quite an accomplishment (Zaher & Unrau, 2007; Bentin, 2011). As optimal as a laboratory setting can be, trying to condense hundreds of millions of years of prebiotic reactions spanning the entire world into a few conical tubes will be time consuming. Another problem to consider is the unlikely initial presence of a template sequence along with the working ribozyme. The ribozyme would either need another copy of itself or its complementary strand in order to replicate itself. Montmorillonite ligation is not specific and would produce random sequence strands of RNA. Chances are low that two identical sequences would be created in this manner. In addition to the ~20 nt extension of general template sequences, Bentin also achieved a 95 nt extension (Bentin, 2011). However, this was only possible with a specific template sequence and did not occur with any other provided templates. It is a very promising step toward full self-replication, but template sequence generality is important in an evolutionary context. The ribosome would be useless if

it could only translate a few genes rather than the entire genome. Because full replicative polymerization has not yet been achieved with B6.61, selection of a better version is not as simple as the in vitro oligonucleotide-ligating ribozymes, where the best come to dominate the population due to their replicative speed and can be selected successively merely through serial transfers to new substrate solutions. The gel extraction method for selection is time consuming in comparison. Conclusion Despite their challenges, ribozymes have made an interesting niche for themselves in the field of abiogenesis. The evolution of a successful RNA polymerase ribozyme is a lofty goal. While its discovery would not be the be-all and end-all of abiogenesis research, it would represent an important stepping stone between prebiotic chemistry and life. The encapsulation of such a ribozyme is also an important step, as it would enable a system of heredity and evolution through natural selection. Based on progress in current research, it is only a matter of time before that ribozyme is discovered. References Athavale, S. S., Petrov, A. S., Hsiao, C., Watkins, D., Prickett, C. D., Gossett, J. J., . . . Williams, L. D. (2012). RNA Folding and Catalysis Mediated by Iron (II). Plos One, 7(5). doi: 10.1371/journal.pone.0038024 Barta, A., Dorner, S., & Polacek, N. (2001). Mechanism of ribosomal peptide bond formation. Science (New York, N.Y.), 291(5502). Basile, B., Lazcano, A., & Oro, J. (1984). Prebiotic syntheses of purines and pyrimidines. Advances in space research : the official journal of the Committee on Space Research (COSPAR), 4(12). doi: 10.1016/0273-1177(84)90554-4 Bentin, T. (2011). A ribozyme transcribed by a ribozyme. Artificial DNA: PNA & XNA, 2(2), 40-42. Callahan, M. P., Smith, K. E., Cleaves, H. J., II, Ruzicka, J., Stern, J. C., Glavin, D. P., . . . Dworkin, J. P. (2011). Carbonaceous meteorites contain a wide range of extraterrestrial nucleobases. Proceedings of the National Academy of Sciences of the United States of America, 108(34). doi: 10.1073/pnas.1106493108 Deamer, D., Dworkin, J. P., Sandford, S. A., Bernstein, M. P., & Allamandola, L. J. (2002). The first cell membranes. Astrobiology, 2(4), 371-381. doi: 10.1089/153110702762470482 Ertem, G. (2004). Montmorillonite, oligonucleotides, RNA and origin of life. Origins of life and evolution of the biosphere : the journal of the International Society for the Study of the Origin of Life, 34(6). doi: 10.1023/B:ORIG.0000043130.49790.a7

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