J Mol Evol (2014) 78:251–262 DOI 10.1007/s00239-014-9623-2

ORIGINAL ARTICLE

Generation of Oligonucleotides Under Hydrothermal Conditions by Non-enzymatic Polymerization Veronica DeGuzman • Wenonah Vercoutere Hossein Shenasa • David Deamer



Received: 29 January 2014 / Accepted: 2 May 2014 / Published online: 13 May 2014 Ó Springer Science+Business Media New York 2014

Abstract We previously reported that 50 -mononucleotides organized within a multilamellar lipid matrix can produce oligomers in the anhydrous phase of hydration– dehydration (HD) cycles. However, hydrolysis of oligomers can occur during hydration, and it is important to better understand the steady state in which ester bond synthesis is balanced by hydrolysis. In order to study condensation products of mononucleotides and hydrolysis of their polymers, we established a simulation of HD cycles that would occur on the early Earth when volcanic land masses emerged from the ocean over 4 billion years ago. At this stage on early Earth, precipitation produced hydrothermal fields characterized by small aqueous pools undergoing evaporation and refilling at elevated temperatures. Here, we confirm that under these conditions, the chemical potential made available by cycles of hydration and dehydration is sufficient to drive synthesis of ester bonds. If 50 -mononucleotides are in solution at millimolar concentrations, then oligomers resembling RNA are synthesized and exist in a steady state with their monomers. Furthermore, if the mononucleotides can form complementary base pairs, then some of the products have properties suggesting that secondary structures are present, including duplex species stabilized by hydrogen bonds. Keywords RNA  Condensation reactions  Hydrolysis  Organizing matrix  Prebiotic chemistry V. DeGuzman  W. Vercoutere Advanced Studies Laboratory, NASA Ames Research Center, Moffett Field, CA, USA H. Shenasa  D. Deamer (&) Department of Biomolecular Engineering, University of California, Santa Cruz, CA 95064, USA e-mail: [email protected]

Introduction The polymeric biomolecules of living cells today exist in a steady state far from equilibrium with energy-dependent synthesis of polymers balancing hydrolysis into monomers. Because the hydrolysis reaction is much slower than synthesis, the polymers exist in a kinetic trap. A similar balance is likely to have prevailed on the prebiotic Earth in which a variety of chemical reactions cycled monomers and their corresponding polymers in steady states, away from thermodynamic equilibrium. The polymeric products of such reactions served as a feed stock for the spontaneous natural experiments and selection of functional polymers that ultimately led to the emergence of living systems. There is a consensus that the earliest forms of cellular life likely used a polymer resembling ribonucleic acid both as a catalyst and as a way to store genetic information (Woese 1967; Crick 1968; Orgel 1968; White 1976; Gilbert 1986; Atkins et al. 2006; Cech 2011; Robertson and Joyce 2012). This conjecture has multiple ways to be tested experimentally, and there has been steady progress since the discovery of ribozymes. For instance, previous investigations demonstrated that functional species of RNA can evolve if cycles of selection and amplification are imposed on mixtures containing random sequences (Ellington and Szostak 1990; Bartel and Szostak 1993). These studies required enzyme-catalyzed reactions, so the results are not directly analogous to prebiotic conditions, but other selective processes related to chemical and physical stability of polymers were likely to be present. For instance, Wo¨chner et al. (2011) and Attwater et al. (2013) used in vitro evolution in ice eutectics to develop a ribozyme polymerase capable of synthesizing RNA strands up to 206 nucleotides in length. Despite this progress, there remains a significant gap in our knowledge: How were mixtures of

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RNA-like molecules originally produced in the absence of catalysts and metabolism? One clue to a possible synthetic pathway was provided by Rajamani et al. (2008) who reported that in the presence of phospholipid as an organizing matrix, 50 -mononucleotides could form RNA-like polymers upon exposure to hydration– dehydration (HD) cycles. It was surmised that the lipid acted as a multilamellar liquid crystalline matrix that organized the monomers within two-dimensional planes between lipid head groups, thereby overcoming entropic barriers (Deamer 2012). This type of ordering which can occur was recently established by an X-ray diffraction investigation of AMP in a multilamellar phospholipid matrix (Toppozini et al. 2013). Since amphiphilic compounds are readily produced by Fischer–Tropsch type synthesis (Rushdi and Simoneit 2001) and are present in carbonaceous meteorites (Deamer and Pashley 1989), it seems reasonable to assume that they were available in the mixture of organic compounds on the prebiotic Earth and could form multilamellar structures upon drying. Even though these conditions are capable of promoting synthesis of ester linkages between mononucleotides, hydrolysis will also occur in the hydrated phase of the cycle. To better understand the steady state in which hydrolysis is balanced by synthesis, we used a model system to investigate the behavior of RNA under conditions simulating the hydrothermal fields that are ubiquitous in today’s volcanic regions, with the assumption that similar conditions would be common on the prebiotic Earth. Hydrothermal fields are characterized by hot springs that supply large and small pools of water at elevated temperature ranges (60–100 °C). They are relatively acidic (pH 2–3) and undergo constant HD cycles as they evaporate and are refilled. In this regard, they are very different from marine hydrothermal vents that have also been proposed as sites for life’s origin (Baross and Hoffman 1985; Martin et al. 2008; Koonin 2007). The main goal of our study here was to investigate how mononucleotides and their polymers behave under such conditions. We were particularly interested in understanding the steady state between synthesis and hydrolysis of phosphodiester bonds in RNA oligomers in the presence and absence of an organizing matrix. We also asked whether oligomers synthesized from mixtures of mononucleotides capable of base pairing had properties related to secondary structure and duplex species. The following specific questions were addressed:

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To what extent does hydrolytic degradation of nucleotide oligomers occur upon exposure to multiple HD cycles at elevated temperatures and acidic pH ranges? Under what conditions can the reaction of ester bond hydrolysis be reversed so that oligomers of mononucleotides are produced by phosphodiester bond synthesis?

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If mononucleotides capable of base pairing are present in a mixture undergoing HD cycles, do their oligomers exhibit secondary structure and possible duplex species?

Methods Reagents We chose 50 adenosine monophosphate (AMP) and 50 uridine monophosphate (UMP) as monomers, and polyadenylic acid (polyA) and polyuridylic acid (polyU) as model polymers, both as single-stranded and duplex forms. The nucleotides and polynucleotides were purchased from Sigma-Aldrich as free acids and used without further purification. As a standard for some of the analytical methods, we synthesized complementary 20mer strands of RNA containing AMP and UMP as monomers (prepared by Protein and Nucleic Acid Facility, Beckman Center, Stanford, CA). The sequences were 50 -AUUAUAAAUUUAUAUUA AUA-30 and 50 -UAUUAAUAUAAAUUUAUAAU-30 and were designed so that potential intramolecular base pairing was minimized. The RNA 20mers could be studied either as single-strands or as duplex species prepared by mixing the two in a 1:1 ratio. The duplex 20mer was sufficiently stable to migrate on a 2 % agarose gel, appearing as a tight fluorescent band after staining with ethidium bromide in the gel. Using an RNA ladder in the same gel as a guide, the band was in the range expected for a 20mer. A previous study (Rajamani et al. 2008) demonstrated that several phospholipids including phosphatidylcholine, phosphatidic acid, and lysophosphatidylcholine (LPC) could promote polymerization of mononucleotides by organizing and concentrating the monomers within the matrix of liquid crystals. For the research reported here, we chose to use 1-palmitoyl, 2-hydroxy lysophosphatidylcholine purchased from Avanti Polar Lipids. Although LPC would not be considered to be prebiotically plausible, it satisfies the minimum experimental requirement for an amphiphilic molecule. LPC is convenient because it forms micelles in dilute solutions, but assembles into membranous multilamellar structures upon drying, thereby avoiding the necessity for preparing liposomes as an intermediate step.

We constructed a chamber that simulated the HD cycles associated with geothermal hot springs and fluctuating pools (Fig. 1). In a typical experiment, glass vials (1.5 mL) containing reaction mixtures to be tested were placed in 24 wells around the perimeter of an aluminum disk within an

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Fig. 1 Hydrothermal field simulation chamber. See text for description

enclosed chamber. The chamber was filled with carbon dioxide, an inert gas, to produce and maintain anaerobic conditions. Carbon dioxide was used rather than nitrogen because it is denser than air. The chamber could be initially filled with gas simply by adding dry ice before the chamber was closed at the start of an experimental run. The amount of dry ice added was twice that calculated to fill the volume of the chamber with CO2 as the solid phase ice sublimed into gas. The disk was heated to a desired temperature, usually 85 °C, and rotation of the disk was controlled by a programmed stepper motor at a rate of 15° every 7.5 min. As the disk rotated, each sample was exposed for 30 min to a flow of dry carbon dioxide through four ports on either side of the disk. Besides maintaining anaerobic conditions, the gas flow also carried away water vapor as it left the reaction mixture. A flow meter set at 4 cu ft per hour controlled the total volume of gas into the chamber, and there was sufficient gas leakage so that a small positive pressure gradient was maintained to prevent room air from entering the chamber. Following dehydration by the gas flow, samples were rehydrated when the rotation brought a vial under each of two ports delivering water from a programmable syringe pump at a rate of 0.1 mL per 7.5 min. The rate of rotation caused each sample to undergo one HD cycle every 90 min. The water evaporating from the samples and then as a product of condensation reactions was absorbed by Drierite (500 g) distributed in twelve open Petri dishes on the floor of the chamber. It was also convenient to carry out smaller scale experiments using glass slides with two wells on each slide that hold 0.1 mL of the reaction mixture. Four slides could be arranged on a laboratory hot plate set at the desired

temperature, and a plastic box with eight small holes (1 mm diameter) was set over the slides. Each hole was placed directly over a well so that carbon dioxide gas flowed into the mixture. A flow meter monitored the gas flow which was set at *2 cc/s per well. Cycles of hydration were performed by briefly removing the box and delivering 50 lL of water to each well. Reaction Mixtures For oligomer synthesis, a typical reaction mixture in the vials undergoing HD cycles in the simulation chamber had 0.2 mL of 10 mM mononucleotides and 0.1 mL of 10 mM LPC. From results of preliminary experiments, the 2:1 mol ratio of mononucleotides to LPC gave the highest yield. Other variables that were optimized included temperature, initial pH, and the number of HD cycles. If the temperature was above 100 °C, then a prominent browning reaction began to occur, while product yields were markedly reduced at temperatures below 70 °C. We therefore chose 85 °C as a standard temperature. The default pH of the mixture was *2.5 because the mononucleotides were present in their acid form, and not as sodium salts. We found that at least four cycles were necessary to produce several micrograms of products for analysis. A second reaction mixture was prepared to investigate hydrolysis. This contained 20 lg of polyA, polyU, or a mixture of the two homopolymers (polyA–polyU) in 0.2 mL water, with the relative concentrations adjusted so that the mole ratio of AMP to UMP was 1:1. The duplex species ranged from 100 to 300 nt when compared to a dsRNA ladder in a gel. The polyA–polyU was used to

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determine whether and to what extent hydrolysis takes place, and as a standard for hyperchromicity studies. A third mixture contained one or both of the synthetic 20mers, 50 -AUUAUAAAUUUAUAUUAAUA-30 and 50 UAUUAAUAUAAAUUUAUAAU-30 , either as singlestranded or as duplex species. These were used as standards for gel electrophoresis and nanopore analysis. Isolation of Products The polymer products were isolated by precipitation in ethanol followed by centrifugation (Rio et al. 2010). The products readily formed pellets, a property consistent with polymers that behaved like RNA. The behavior was confirmed by testing whether the polymers could be isolated with spin tubes designed to purify RNA from mixtures (Life Technologies), and yields similar to those obtained by ethanol precipitation were obtained. However, ethanol precipitation tended to give a greater yield of products, so it was the method of choice. This protocol requires addition of a salt such as sodium or ammonium acetate. We tested both salts and found that ammonium acetate tended to enhance staining of products by ethidium bromide in an agarose gel; so in a typical procedure, ‘ volume of 5.0 M ammonium acetate, pH 5.8, was added to a sample to be precipitated. This was followed by adding three volumes of 100 % ethanol, cooling to -20 °C for 15 min, and centrifugation at 14,000 rcf for 30 min. The pellet was rinsed once with 70 % ethanol, then resuspended in 50 lL of deionized water. Gel Electrophoresis In order to probe for possible secondary structures and duplex species, precast 4 % agarose gels (Invitrogen) were used to visualize polymeric products. The gels contained ethidium bromide dye which displays enhanced fluorescence upon intercalation between stacked bases of duplex nucleic acid species. To check whether the fluorescence was an artifact of ethidium staining, products were also analyzed in precast 2 % agarose gels containing SYBRSafe dye which also stains by intercalation.

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heated in a cuvette with a circulating water bath, and absorbance was monitored as the temperature increased from 15 to 75 °C. Nanopore Analysis Products were analyzed by a single-molecule nanopore technique. The method is described in detail in Akeson et al. (1999). Briefly, a single a-hemolysin channel was inserted into a 30-lm diameter planar lipid bilayer of diphytanoylphosphatidylcholine (Avanti Polar Lipids), and a voltage of 180 mV was imposed. We used a solution of 1.0 M KCl buffered at pH 7.5 with HEPES, so the voltage drove an ionic current of *200 pA through the open channel. The limiting aperture of the hemolysin channel has a diameter of 1.5 nm, a size that allows only singlestranded nucleic acid molecules to pass through. From the previous work (Kasianowicz et al. 1996; Akeson et al. 1999), it is known that a single-stranded RNA such as poly(A) or poly(U) passing through the channel produces a characteristic blockade of ionic current that reduces the open-channel current by *85 to 90 %. The residence time of single-stranded oligomers translocating through the pore is roughly related to the length of the strand. Earlier studies also determined that double-stranded DNA or RNA molecules can be captured in the larger diameter entry vestibule of the channel but with helical diameters of 2.0 or 2.3 nm, molecules are unable to traverse the limiting aperture (Kasianowicz et al. 1996). However, short DNA hairpins produce a partial ionic current blockade when the duplex stem enters the vestibule of the channel, with an amplitude dependent on the number of base pairs in the stem (Vercoutere et al. 2001). If the double-stranded stem of the hairpin is in the range of 3–10 base pairs, then it can spontaneously unzip following a stochastic duration of the partial blockade, and the single-stranded molecule is then translocated through the limiting aperture, producing a characteristic 85–90 % spike at the end of the blockade signal. The characteristic blockade signals can be interpreted in terms of the structure and dynamic motion of the molecules, and we used this information in the present study to interpret nanopore signals produced by polymerization products.

Spectrophotometry Product yields were determined by NanoDrop spectrophotometry (Thermo Scientific). We used hyperchromicity measurements to test whether the products had secondary structure or duplex species that would exhibit a temperature-dependent increase in absorbance at 260 nm. For this purpose, sufficient amounts of products were dissolved in 25 mM phosphate buffer, pH 7.0, to give an initial absorbance value around 0.4 units. One mL of the sample was

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Results Stability of RNA Under Simulation Conditions It was essential to establish the degree to which a known RNA sample undergoes hydrolysis during HD cycles at acidic pH ranges and elevated temperatures. For instance, if RNA hydrolyzed completely in a single cycle, then there

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70 lg of the product was used in this gel in order to reveal the full extent of the product band, while just 2 lg of the polyA–polyU duplex was required to produce the more prominent band shown in the gel. Figure 3b shows *16 lg of product in a 2 % agarose gel stained with SYBR-Safe. The product has traveled farther in 2 % agarose and forms a more condensed band that is stained by the intercalating dye. The lighter streak above the product band is caused a small amount of browning product produced during cycling which absorbs the blue illumination used to excite SYBRSafe fluorescence. The band produced by the synthetic dsRNA 20mer is also stained by SYBR-Safe. Cycling Markedly Enhances Yields of Oligomeric Products

Fig. 2 Relative stability of polyA–polyU. An example gel is illustrated. The gel shows duplicate samples of polyA–polyU exposed to four sequential 30-min HD cycles at 85 °C, pH 3. The graph shows mean and SD of four separate experiments in which 20 lg polyA– polyU was exposed to four HD cycles, then precipitated in ethanol. The Y-axis shows the percent of polymer remaining after each cycle

would be no reason to expect significant synthesis of oligomers. Figure 2 shows a gel of the polyA–polyU duplex going through four HD cycles, and a graph of the amount remaining. On average, approximately 8 % of the duplex polymer was lost after each cycle. Condensation Reactions Produce RNA-Like Oligomers: Intercalating Dyes It is well known that the fluorescence of certain dyes is markedly enhanced when they intercalate into doublestranded polynucleotides. This effect is illustrated in Fig. 3a, which shows a gel with polyA, polyU, and the duplex species polyA–polyU produced by mixing the two homopolymers in a 1:1 mol ratio of A:U. The homopolymers do not bind the ethidium bromide dye in the gel, but mixing the polyA and polyU to produce an equal amount of the duplex species polyA–polyU gives a strongly fluorescent band. This is an important point, because dye intercalation can be used as an indicator of possible duplex structures in polymerization products. Figure 3a also shows typical products of an experimental reaction of the 50 mononucleotides AMP and UMP exposed to six HD cycles. The products bind the dye, suggesting that a limited amount of duplex structures might be present. However, it is likely that only a fraction of the products have hydrogenbonded secondary structures. For purposes of illustration,

Figure 4 shows typical yields of oligomers relative to the number of cycles. Although the amount of synthesis varied from one run to the next, the general trend was toward increasing amounts of a product with additional cycles, followed by a plateau after 4 cycles. We assume that the plateau reflects the point at which condensation into oligomers is balanced by the back reaction of hydrolysis. The back reaction would also limit the length of the chains, because hydrolysis occurs randomly. This means that longer chains are more likely to have a single break in the strand than shorter chains, thereby maintaining a steady state of shorter fragments. Hyperchromicity To determine whether products displayed hyperchromicity, a sufficient amount of each sample was added to 1.0 mL of 25 mM sodium phosphate buffer, pH 7.0, to give an absorbance between 0.2 and 0.4 at 260 nm. The absorbance was then monitored while heating from 15 to 70 °C in a temperature-controlled cuvette. Figure 5a shows the hyperchromicity of a known duplex RNA composed of polyA mixed in a 1:1 A:U ratio with polyU, and Fig. 5b shows results for products synthesized from a 1:1 mix of AMP and UMP, or AMP alone. The hyperchromicity exhibited by the polyA–polyU is that expected from a mixture of duplex strands 100–200 nucleotides in length (Doty et al. 1959). The oligomers produced from 1:1 mixtures of AMP and UMP showed obvious hyperchromicity that increased with temperature, while the polymeric product of AMP by itself had no indication of hyperchromicity. The hyperchromicity of products varied considerably from one run to the next. The result shown in Fig. 5b is the largest seen in 10 separate runs, but more commonly the hyperchromicity is from *5 to 15 %. Two such results are shown in Fig. 5c in which the increments in absorbance are normalized against the initial absorbance. After cooling for

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Fig. 3 a Precast gel (Invitrogen, 4 % agarose, ethidium bromide staining) showing polyU, polyA, and polyA–polyU. Only the duplex polymer is stained by ethidium bromide, an intercalating dye. The first lane on left is a ssRNA ladder with the 100mer marked, and the

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last lane on the right shows that the product from an experimental HD reaction of AMP ? UMP ? lipid is also stained by the ethidium dye. b Precast gel (Invitrogen, 2 % agarose, SYBR-Safe stain)

remained near its peak value. This suggested to us that precipitation in ethanol and ammonium acetate produced concentrated pellets in which hydrogen bonding between polymer strands was maximized, thereby promoting secondary structures and duplex species. When the concentrated samples were dissolved in buffer for hyperchromicity determination, the secondary structure melted as temperature increased, but the dilute random sequences could not then readily reanneal upon cooling. Nanopore Analysis of Products

Fig. 4 Polymer yields increased with the number of cycles when a 1:1 mixture of 50 -AMP and 50 -UMP (5 mM each) and 5 mM LPC was exposed to multiple 30 min HD cycles, 85 °C, starting pH 2.5. The products were precipitated in ethanol, then dissolved in 50 lL water, and analyzed by absorbance at 262 nm with a NanoDrop instrument. Each point is the yield of product (mean and standard deviation) in four sample vials from each of six cycles. The 0 cycle was obtained in the same way using four samples that were not exposed to cycling. The blank (0 cycle) mean value was 0.31 with an SD of 0.14, and was subtracted from the mean values of the cycled samples

several hours, the hyperchromicity of the polyA–polyU returned to the original value, as expected. However, in the same time interval, the hyperchromicity of HD products

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When linear anionic polymers such as polynucleotides are captured by the electric field in a nanopore, they are drawn through a single-molecule electrophoresis. As described in Methods, the pore has a limiting aperture of *1.5 nm, so a single-stranded nucleic acid, therefore, blocks much of the ionic current as it is translocated from the cis to the trans side of the membrane. Modulation of the blockade signals provides information about the structure and dynamic motion of the polymer during translocation. Preliminary results from gel analysis suggested that the majority of our products ranged from 20 to 50 nt in length. Using this as a guide, we synthesized an RNA 20mer to be used as a standard, both in the gels and for nanopore analysis. Figure 6a, b shows event diagrams and sample blockades for the control single-stranded and double-

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the event diagram represents a single molecule interacting with the pore to produce an ionic current blockade having a characteristic amplitude (Y-axis) and duration (X-axis). (See ‘‘Discussion’’ for detailed interpretation.) In Fig. 6a, typical blockades produced by the ss20mer ranged from 0.2 to 5 ms in duration and had amplitudes between 50 and 75 % of the open-channel current, with most less than a millisecond duration. The blockade signatures were often characterized by a partial blockade followed by a rapid downward spike. The partial blockade is produced when the oligomer transiently remains in the vestibule of the hemolysin channel, and the spike occurs when the molecule moves through the limiting aperture to produce a full blockade. The less common longer blockades were in the range of several milliseconds duration and approximately 80–90 % reduction of the open-channel current. Figure 6b shows an event diagram for the control double-stranded RNA 20mer. The most common blockades were again less than a millisecond in duration, with a trend toward deeper amplitudes than the ssRNA 20mer. Individual blockades exhibited downward spikes resembling those of ssRNA 20mers but with a somewhat greater average amplitude. There was a trail of deeper blockades with durations of 10 ms and longer that also tend to have exit spikes. Figure 6c shows an event diagram for the polymer synthesized from AMP and UMP with LPC present as an organizing matrix. The pattern of blockades was distinctly different from the ss and dsRNA 20mers, with the majority of events in a group having 25 % amplitude (*-50 DpA) and durations from 0.5 to 10 ms. About 10 % of the events had deeper amplitudes and similar durations. These tended to be noisier than the 20mers and rarely showed exit spikes. A detailed discussion and interpretation of the blockade signals follows.

Discussion Fig. 5 Hyperchromicity of poly A–polyU and products of AMP and UMP HD-driven polymerization. a PolyA–polyU showed an extended hyperchromicity effect between 20 and 70 °C, as expected for a duplex species with variable chain lengths [100 nt. b The product of AMP and UMP HD-driven polymerization exhibited hyperchromicity between 20 and 40 °C, but the product of AMP alone did not exhibit hyperchromicity (lower trace). c Typical hyperchromicity of two different reaction products normalized by taking the initial absorbance to be 100

stranded synthetic RNA 20mer, respectively, while Fig. 6c shows the event diagram and sample blockades for the products of HD-cycled AMP ? UMP ? LPC. Each dot in

The results reported here support the following conclusions: 1.

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A 1:1 mixture of two mononucleotides capable of base pairing—50 -AMP and 50 -UMP—undergoes polymerization when exposed to multiple HD cycles at acidic pH, confirming the observations of Rajamani et al. (2008). We also demonstrated that polyA–polyU, a model system of duplex oligomers, was only slowly hydrolyzed under the conditions used to drive polymerization. The oligomers have lengths ranging from *10 to [50 nucleotides when analyzed by gel electrophoresis on 4 % agarose.

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Fig. 6 Event diagrams and example blockade patterns for control ss and ds 20mer RNA molecules, and for HD-cycled AMP ? UMP ? lipid product. Note that amplitude scale is the same, while duration scale is different for each type of molecule examined. a The event diagram of ssRNA 20mers shows amplitude and duration of ionic current blockades when a 20mer is captured by the nanopore. Each dot represents a single molecule interacting with the nanopore.

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Individual blockades of shorter and longer events are shown on the right. b The event diagram for double-stranded RNA 20mer also reveals short and long blockades, with examples of individual events shown on the right. c The event diagram for products of HD-cycled AMP ? UMP ? LPC reveals a majority of short duration events and fewer long events than the control 20mers

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3.

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The oligomers are stained in gels containing the intercalating dyes ethidium bromide and SYBR-Safe, indicating that a certain amount of hydrogen-bonded secondary structures is present in the products. The oligomers exhibit hyperchromicity, consistent with the presence of duplex structures stabilized by hydrogen bonding between base pairs. Nanopore analysis confirms that oligomers are synthesized when AMP and UMP undergo HD cycles. Some of the blockade signals are characteristic of linear anionic species that translocate through the pore, but the majority are short-lived partial blockades that have not been previously reported.

Analysis of Products The first goal in the research reported here was to determine whether the plausible prebiotic conditions involving HD cycles at elevated temperatures can produce mixtures of RNA-like polymers. A second goal was to determine whether the products of such a reaction were sufficiently stable to remain in a steady state with monomers. It is important to note that the procedures used for biological RNA analysis cannot be expected to work ideally on the products of HD-driven polymerization. The reason is that the products are mixtures of hundreds if not thousands of different linear molecules containing random sequences of AMP and UMP linked by 20 –50 and 30 –50 bonds. The molecules have varying lengths and degrees of intramolecular folding, as well as partial duplex structures between molecules. We found that the usual analytical methods of HPLC cannot separate the components of such a mixture, nor can they be analyzed by MALDI or ESI mass spectrometry because only nanogram amounts of individual products are present in a few micrograms of total yield. However, we were able to use gel electrophoresis as a rough guide to strand lengths, and intercalating dyes to probe for possible duplex structures. Spectrophotometry at 260 nm gave approximate values for the amounts of products, and nanopore analysis with a single-molecule resolution confirmed that polymers were present. Hydrolytic Degradation of Nucleotide Oligomers When RNA is exposed to HD cycles in the simulation chamber, drying of the 0.2 mL reaction mixture occurs within 2–3 min at 85 °C followed by dehydration for another 60 min before the next rehydration occurs. There have been no previous reports of RNA exposed to these conditions, so it was essential to establish stability. The results in Fig. 2 make it clear that RNA is relatively resistant to hydrolysis, even when undergoing multiple HD

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cycles at 85 °C at acidic pH. Just a few percent of the total RNA is lost in each cycle as estimated from the amounts precipitated in ethanol. Although the polyA–polyU was added as the duplex species, the strands will dissociate at the elevated temperature, so it is actually single-stranded RNA that undergoes hydrolysis, then reassembles upon cooling. These results are consistent with those of Kawamura (2004) who reported that the half-life of oligoribonucleotides 11, 17, 18, and 23 nt in length was approximately an hour at 100 °C. The reaction was carried out in aqueous salt solutions (0.1 M NaCl and MgCl2) buffered at pH 8, while our HD cycles were at 85 °C and pH 3. Furthermore, our solutions were only hydrated for a few minutes before becoming completely dry at the elevated temperature, so hydrolysis would be less extensive in comparison to the hour-long incubations described by Kawamura. Synthesis of Oligomers During HD Cycles Three lines of evidence confirm the presence of polymers synthesized by condensation reactions in an organizing lipid matrix. First, electrophoresis in agarose gels shows a main product band between 10 and [50 nt in length, the same range reported by Rajamani et al. (2008) for products labeled with 32P. Second, the products can be precipitated by a standard ethanol method used to isolate RNA, and yields depend on the number of HD cycles. Finally, the products can be detected by the blockades, they produce when analyzed by a nanopore technique. The condensation reaction is best understood as an acid catalyzed ester bond formation. We are extrapolating from the well-understood mechanism for synthesis of carboxylate esters, because to our knowledge, there have been no previous studies of phosphate ester synthesis driven by dehydration. When a proton binds to an acid group such as carboxylate or phosphate, a nucleophilic center is produced that can be attacked by the hydroxyl group of the second reactant, in this case ribose, followed by rearrangement of bonds so that water becomes a leaving group. In a sense, this is simply the reversal of a hydrolysis reaction in which water is added to an ester bond. The chemical potential and activation energy driving polymerization are provided by dehydration at elevated temperature ranges. The reactants are concentrated during dehydration, then organized between lipid lamellae in such a way that ester bond synthesis is promoted. If the rate of synthesis exceeds the rate of hydrolysis, then it is inevitable that polymers will accumulate in a kinetic trap during the first few cycles of HD, then reach a steady state in later cycles when the rate of hydrolysis of longer strands balances the rate of synthesis.

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Fig. 7 Interpretation of nanopore blockade signatures. See text for details

Interpretation of Nanopore Results The complexity of the blockade signals produced by ssRNA 20mers and dsRNA 20mers was surprising. Much longer homopolymers of DNA and RNA translocate at rates in which each base passes through the pore in times averaging about 1–2 ls per base for pyrimidine nucleotides such as polyU or polyC (Meller et al. 2000). Thus, a 20mer should on average translocate in *20 to 40 ls; yet, the majority of blockades produced by the synthetic 20mers had longer durations ranging from 100 ls to several milliseconds. Our working hypothesis is that a short and highly flexible ssRNA 20mer can become tangled in the

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vestibule of the hemolysin channel, and translocation is then delayed until it untangles sufficiently to pass through the limiting aperture of 1.5 nm diameter (Fig. 7a). This produces a partial blockade followed by a spike (Fig. 6a). The tangled coil also has the potential to escape back into the cis side medium, in which case only a partial blockade is produced without an exit spike. On the other hand, it is known that duplex species of DNA in the form of hairpins cannot immediately penetrate the limiting aperture of the hemolysin pore. Instead, the duplex end of a hairpin is captured in the vestibule, resulting in a partial blockade, then unzips and passes through as a single strand that produces an exit spike

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(Vercoutere et al. 2001). We speculate that the dsRNA 20mers may go through a similar process of capture and unzipping, and thereby produce blockades with exit spikes shown in Fig. 7 and illustrated in Fig. 6b. The polymers produced by condensation reactions of AMP and UMP are much more complex than the pure RNA 20mers. Gel electrophoresis reveals varying strand lengths, presumably linked by 20 –50 and 30 –50 phosphodiester bonds. Single-strands and secondary structures such as coils and duplex species are likely to be present. We assume that these oligomers will interact with the pore in a variety of ways both similar to and different from those illustrated for the ss and dsRNA 20mers. Some will be drawn partially into the vestibule (Fig. 7c), producing a low amplitude blockade but then escaping. Others, both single and double stranded, will form tangles in the vestibule, producing the noisy longer signals shown in Fig. 6c when single-stranded ends transiently enter and come out of the limiting aperture. The molecule will either become sufficiently untangled to translocate or will escape. Although a complete understanding will require further study, the fact that blockades are produced confirms that polymers have been synthesized and produce a variety of complex signals when they interact with the nanopore. Nature of Products: Alternative Interpretations In the study reported by Rajamani et al. (2008), most of the work was done with a single nucleotide present, such as AMP or UMP. The products were end-labeled with 32P, and presumably consisted of oligonucleotides ranging from 20 to 100mers. Although it might at first seem equally simple to describe the products of a random condensation reaction involving two monomers, in fact, the mixture of products may become much more complex because of the potential for hydrogen-bonded secondary structures to emerge. The complexity is further increased by the fact that all of the species can have lengths varying from a few nucleotides up to 50 or more according to the distribution seen in the gels. An additional complication is that both 20 – 50 and 30 –50 bonds can form. A similar distribution of complex products would characterize any polymerization reaction that could occur in the prebiotic environment. Two lines of evidence suggest that the oligomer products have a certain degree of secondary structure. Intercalating dyes stain the product strands in agarose gels but do not stain single-stranded homopolymers of polyA or polyU. Second, the products show clear evidence of hyperchromicity, with a transition temperature between 20 and 40 °C. In previous studies, this temperature is typical of duplex strands composed of mixed AMP and UMP between 10 and 15 base pairs long (Borer et al. 1974). The possible presence of secondary structures in random polymers composed of AMP and UMP

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is not surprising. It has long been known from hyperchromicity results that mRNA and synthetic RNA undergo a remarkable degree of folding stabilized by hydrogen bonding (Doty et al. 1959). Gralla and Delisi (1974) used computational modeling to predict that random sequences of RNA will contain as much as 50 % base pairing. This fraction should be even greater when only two bases are present, because of the increased likelihood of finding favorable sequences of complementary pairs. Although the evidence is consistent with some of the products being linear polymers having a certain degree of hydrogen-bonded secondary structure, other possibilities must be taken into account. For instance, the majority of blockades shown in Fig. 6c does not reflect translocation, but instead appear to be oligomers that partially enter the pore and then escape. This could result from branching linkages in the molecules that inhibit translocation through the limiting aperture. It follows that the polymers are not purely linear species like the synthetic single- and doublestranded synthetic 20mers, but instead are mixtures containing linear oligomers capable of translocation and oligomers with other linkages. Both components would migrate during gel electrophoresis, but only the linear molecules could produce full blockades, or exit spikes, characteristic of translocating oligomers. Although staining with intercalating dyes and hyperchromicity suggests that secondary structure is present in the products, it does not dominate the mixture. For instance, in comparison with the known duplex polyA– polyU in gels, the products are not as strongly stained by intercalating dyes. Furthermore, their typical hyperchromicity is considerably less than that of the polyA–polyU duplex. The apparent secondary structure is intriguing, but further investigation will be needed to establish the extent to which it occurs and its significance. Limitations of Hydrothermal Condensation Reactions There are limitations to any polymerization process proposed to occur in hydrothermal conditions. First, the monomers themselves are only transiently stable at elevated temperatures in acidic pH ranges. For instance, the bond between adenine and ribose in AMP can be broken by depurination, and cytosine can undergo deamination to form uracil. Furthermore, carbohydrates such as ribose are subject to browning reactions that will also reduce its availability as a potential reactant. This means that even if mononucleotides are somehow synthesized, they will only be available for polymerization reactions for limited amounts of time. Another problem is regiospecificity. When ribonucleotides form ester linkages by a non-enzymatic reaction, the bonds can be either 20 –50 or 30 –50 randomly spaced within the polymer strands. Furthermore, organic compounds on

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the prebiotic Earth would be complex mixtures of thousands of racemic compounds. How could specific monomers be sorted out in order to undergo polymerization? Random polymers might be synthesized, but they would be unable to fold consistently into functional structures such as ribozymes. Although these are challenging questions, laboratory simulations of hydrothermal fields provide a model system for addressing them experimentally. Implications for Origin of Life The HD cycles used in this study simulate fluctuating lacustrine environments associated with volcanism, particularly small pools of standing water filled by hot springs and precipitation (Mulkidjaniana et al. 2012). The edges of such pools and the pools themselves continuously undergo cycles of hydration and dehydration. The results reported here support the conclusion that under these conditions, polymers resembling nucleic acids can be synthesized in the absence of enzymes or activated substrates and have the potential to fold into more complex secondary structures. The implication is that similar reactions could give rise to prebiotic polymers. The obvious question that follows is how selection for functional systems of polymers could be initiated. Given that RNA-like molecules and amphiphilic molecules were present on the early Earth, it is plausible that random mixtures were encapsulated in self-assembled boundary membranes. A simple calculation shows that a milligram of membraneforming lipid mixed with random polymers will form trillions of lipid vesicles, each different in composition from all the rest. The compartments and their contents represent microscopic experiments in a natural version of combinatorial chemistry in which selective processes required for evolution can begin (Adamala and Szostak 2013). Acknowledgments This investigation was supported by a Lonsdale Research Award, funded by a generous gift from Harry Lonsdale. Conflict of interest Authors declare that they have no conflict of interest.

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Generation of oligonucleotides under hydrothermal conditions by non-enzymatic polymerization.

We previously reported that 5'-mononucleotides organized within a multilamellar lipid matrix can produce oligomers in the anhydrous phase of hydration...
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