A Prebiotic Genetic Nucleotide as an Early Darwinian Ancestor for Pre-RNA Evolution.

Prebiotic genetic nucleotides (PGNs) often outcompete canonical alphabets in the formation of nucleotides and subsequent RNA oligomerization under early Earth conditions. This indicates that the early genetic code might have been dominated by pre-RNA that contained PGNs for information transfer and catalysis. Despite this, deciphering pre-RNAs' capacity to acquire function and delineating their evolutionary transition to a canonical RNA World has remained under-researched in the origins of life (OoL) field. We report the synthesis of a prebiotically relevant nucleotide (BaTP) containing the noncanonical nucleobase barbituric acid. We demonstrate the first instance of its enzymatic incorporation into an RNA, using a T7 RNA polymerase. BaTP's incorporation into baby spinach aptamer allowed it to retain its overall secondary structure and function. Finally, we also demonstrate faithful transfer of information from the pre-RNA-containing BaTP to DNA, using a high-fidelity RNA-dependent DNA polymerase, alluding to how selection pressures and complexities could have ensued during the molecular evolution of the early genetic code.

Nonenzymatic RNA replication in a mixture of 'spent' nucleotides.

Nonenzymatic template-directed replication would have been affected by co-solutes in a heterogeneous prebiotic soup due to lack of enzymatic machinery. Unlike in contemporary biology, these reactions use chemically activated nucleotides, which undergo rapid hydrolysis forming nucleoside monophosphates ('spent' monomers). These co-solutes cannot extend the primer but continue to base pair with the template, thereby interfering with replication. We, therefore, aimed to understand how a mixture of 'spent' ribonucleotides would affect nonenzymatic replication. We observed the inhibition of replication in the mixture, wherein the predominant contribution came from the cognate Watson-Crick monomer, showing potential sequence dependence. Our study highlights how nonenzymatic RNA replication would have been directly affected by co-solutes, with ramifications for the emergence of functional polymers in an RNA World.

Nonenzymatic Template-Directed Primer Extension Using 2'-3' Cyclic Nucleotides Under Wet-Dry Cycles.

RNA World Hypothesis is centred around the idea of a period in the early history of life's origin, wherein nonenzymatic oligomerization and replication of RNA resulted in functional ribozymes. Previous studies in this endeavour have demonstrated template-directed primer extension using chemically modified nucleotides and primers. Nonetheless, similar studies that used non-activated nucleotides led to the formation of RNA only with abasic sites. In this study, we report template-directed primer extension with prebiotically relevant cyclic nucleotides, under dehydration-rehydration (DH-RH) cycles occurring at high temperature (90 °C) and alkaline conditions (pH 8). 2'-3' cyclic nucleoside monophosphates (cNMP) resulted in primer extension, while 3'-5' cNMP failed to do so. Intact extension of up to two nucleotide additions was observed with both canonical hydroxy-terminated (OH-primer) and activated amino-terminated (NH2-primer) primers. We demonstrate primer extension reactions using both purine and pyrimidine 2'-3' cNMPs, with higher product yield observed during cAMP additions. Further, the presence of lipid was observed to significantly enhance the extended product in cCMP reactions. In all, our study provides a proof-of-concept for nonenzymatic primer extension of RNA, using intrinsically activated prebiotically relevant cyclic nucleotides as monomers.

Distinguishing Biotic vs. Abiotic Origins of 'Bio'signatures: Clues from Messy Prebiotic Chemistry for Detection of Life in the Universe.

It is not a stretch to say that the search for extraterrestrial life is possibly the biggest of the cosmic endeavors that humankind has embarked upon. With the continued discovery of several Earth-like exoplanets, the hope of detecting potential biosignatures is multiplying amongst researchers in the astrobiology community. However, to be able to discern these signatures as being truly of biological origin, we also need to consider their probable abiotic origin. The field of prebiotic chemistry, which is aimed at understanding enzyme-free chemical syntheses of biologically relevant molecules, could particularly aid in this regard. Specifically, certain peculiar characteristics of prebiotically pertinent messy chemical reactions, including diverse and racemic product yields and lower synthesis efficiencies, can be utilized in analyzing whether a perceived 'signature of life' could possibly have chemical origins. The knowledge gathered from understanding the transition from chemistry to biology during the origin of life could be used for creating a library of abiotically synthesized biologically relevant organic molecules. This can then be employed in designing, standardizing, and testing mission-specific instruments/analysis systems, while also enabling the effective targeting of exoplanets with potentially 'ongoing' molecular evolutionary processes for robust detection of life in future explorative endeavors.

Metals in Prebiotic Catalysis: A Possible Evolutionary Pathway for the Emergence of Metalloproteins.

Proteinaceous catalysts found in extant biology are products of life that were potentially derived through prolonged periods of evolution. Given their complexity, it is reasonable to assume that they were not accessible to prebiotic chemistry as such. Nevertheless, the dependence of many enzymes on metal ions or metal-ligand cores suggests that catalysis relevant to biology could also be possible with just the metal centers. Given their availability on the Hadean/Archean Earth, it is fair to conjecture that metal ions could have constituted the first forms of catalysts. A slow increase of complexity that was facilitated through the provision of organic ligands and amino acids/peptides possibly allowed for further evolution and diversification, eventually demarcating them into specific functions. Herein, we summarize some key experimental developments and observations that support the possible roles of metal catalysts in shaping the origins of life. Further, we also discuss how they could have evolved into modern-day enzymes, with some suggestions for what could be the imminent next steps that researchers can pursue, to delineate the putative sequence of catalyst evolution during the early stages of life.

pH-Responsive Self-Assembled Compartments as Tuneable Model Protocellular Membrane Systems.

Prebiotically plausible single-chain amphiphiles are enticing as model protocellular compartments to study the emergence of cellular life, owing to their self-assembling properties. Here, we investigated the self-assembly behaviour of mono-N-dodecyl phosphate (DDP) and mixed systems of DDP with 1-dodecanol (DDOH) at varying pH conditions. Membranes composed of DDP showed pH-responsive vesicle formation in a wide range of pH with a low critical bilayer concentration (CBC). Further, the addition of DDOH to DDP membrane system enhanced vesicle formation and stability in alkaline pH regimes. We also compared the high-temperature behaviour of DDP and DDP:DDOH membranes with conventional fatty acid membranes. Both, DDP and DDP:DDOH mixed membranes possess packing that is similar to decanoic acid membrane. However, the micropolarity of these systems is similar to phospholipid membranes. Finally, the pH-dependent modulation of different phospholipid membranes doped with DDP was also demonstrated to engineer tuneable membranes with potential translational implications.

Porphyrin in Prebiotic Catalysis: Ascertaining a Route for the Emergence of Early Metalloporphyrins.

Metal ions are known to catalyze certain prebiotic reactions. However, the transition from metal ions to extant metalloenzymes remains unclear. Porphyrins are found ubiquitously in the catalytic core of many ancient metalloenzymes. In this study, we evaluated the influence of porphyrin-based organic scaffold, on the catalysis, emergence and putative molecular evolution of prebiotic metalloporphyrins. We studied the effect of porphyrins on the transition metal ion-mediated oxidation of hydroquinone (HQ). We report a change in the catalytic activity of the metal ions in the presence of porphyrin. This was observed to be facilitated by the coordination between metal ions and porphyrins or by the formation of non-coordinated complexes. The metal-porphyrin complexes also oxidized NADH, underscoring its versatility at oxidizing more than one substrate. Our study highlights the selective advantage that some of the metal ions would have had in the presence of porphyrin, underscoring their role in shaping the evolution of protometalloenzymes.

Elucidating N-acyl amino acids as a model protoamphiphilic system.

Protoamphiphiles are prebiotically-plausible moieties that would have constituted protocell membranes on early Earth. Although prebiotic soup would have contained a diverse set of amphiphiles capable of generating protocell membranes, earlier studies were mainly limited to fatty acid-based systems. Herein, we characterize N-acyl amino acids (NAAs) as a model protoamphiphilic system. To the best of our knowledge, we report a new abiotic route in this study for their synthesis under wet-dry cycles from amino acids and monoglycerides via an ester-amide exchange process. We also demonstrate how N-oleoyl glycine (NOG, a representative NAA) results in vesicle formation over a broad pH range when blended with a monoglyceride or a fatty acid. Notably, NOG also acts as a substrate for peptide synthesis under wet-dry cycles, generating different lipopeptides. Overall, our study establishes NAAs as a promising protoamphiphilic system, and highlights their significance in generating robust and functional protocell membranes on primitive Earth.

Influence of Metal Ions on Model Protoamphiphilic Vesicular Systems: Insights from Laboratory and Analogue Studies.

Metal ions strongly affect the self-assembly and stability of membranes composed of prebiotically relevant amphiphiles (protoamphiphiles). Therefore, evaluating the behavior of such amphiphiles in the presence of ions is a crucial step towards assessing their potential as model protocell compartments. We have recently reported vesicle formation by N-acyl amino acids (NAAs), an interesting class of protoamphiphiles containing an amino acid linked to a fatty acid via an amide linkage. Herein, we explore the effect of ions on the self-assembly and stability of model N-oleoyl glycine (NOG)-based membranes. Microscopic analysis showed that the blended membranes of NOG and Glycerol 1-monooleate (GMO) were more stable than pure NOG vesicles, both in the presence of monovalent and divalent cations, with the overall vesicle stability being 100-fold higher in the presence of a monovalent cation. Furthermore, both pure NOG and NOG + GMO mixed systems were able to self-assemble into vesicles in natural water samples containing multiple ions that were collected from active hot spring sites. Our study reveals that several aspects of the metal ion stability of NAA-based membranes are comparable to those of fatty acid-based systems, while also confirming the robustness of compositionally heterogeneous membranes towards high metal ion concentrations. Pertinently, the vesicle formation by NAA-based systems in terrestrial hot spring samples indicates the conduciveness of these low ionic strength freshwater systems for facilitating prebiotic membrane-assembly processes. This further highlights their potential to serve as a plausible niche for the emergence of cellular life on the early Earth.

Spontaneous emergence of membrane-forming protoamphiphiles from a lipid-amino acid mixture under wet-dry cycles.

Dynamic interplay between peptide synthesis and membrane assembly would have been crucial for the emergence of protocells on the prebiotic Earth. However, the effect of membrane-forming amphiphiles on peptide synthesis, under prebiotically plausible conditions, remains relatively unexplored. Here we discern the effect of a phospholipid on peptide synthesis using a non-activated amino acid, under wet-dry cycles. We report two competing processes simultaneously forming peptides and N-acyl amino acids (NAAs) in a single-pot reaction from a common set of reactants. NAA synthesis occurs via an ester-amide exchange, which is the first demonstration of this phenomenon in a lipid-amino acid system. Furthermore, NAAs self-assemble into vesicles at acidic pH, signifying their ability to form protocellular membranes under acidic geothermal conditions. Our work highlights the importance of exploring the co-evolutionary interactions between membrane assembly and peptide synthesis, having implications for the emergence of hitherto uncharacterized compounds of unknown prebiotic relevance.

Prebiological Membranes and Their Role in the Emergence of Early Cellular Life.

Membrane compartmentalization is a fundamental feature of contemporary cellular life. Given this, it is rational to assume that at some stage in the early origins of life, membrane compartments would have potentially emerged to form a dynamic semipermeable barrier in primitive cells (protocells), protecting them from their surrounding environment. It is thought that such prebiological membranes would likely have played a crucial role in the emergence and evolution of life on the early Earth. Extant biological membranes are highly organized and complex, which is a consequence of a protracted evolutionary history. On the other hand, prebiotic membrane assemblies, which are thought to have preceded sophisticated contemporary membranes, are hypothesized to have been relatively simple and composed of single chain amphiphiles. Recent studies indicate that the evolution of prebiotic membranes potentially resulted from interactions between the membrane and its physicochemical environment. These studies have also speculated on the origin, composition, function and influence of environmental conditions on protocellular membranes as the niche parameters would have directly influenced their composition and biophysical properties. Nonetheless, the evolutionary pathways involved in the transition from prebiological membranes to contemporary membranes are largely unknown. This review critically evaluates existing research on prebiotic membranes in terms of their probable origin, composition, energetics, function and evolution. Notably, we outline new approaches that can further our understanding about how prebiotic membranes might have evolved in response to relevant physicochemical parameters that would have acted as pertinent selection pressures on the early Earth.

Emergence of ribozyme and tRNA-like structures from mineral-rich muddy pools on prebiotic earth.

The RNA world hypothesis, although a viable one regarding the origin of life on earth, has so far failed to provide a compelling explanation for the synthesis of RNA enzymes from free nucleotides via abiotic processes. To tackle this long-standing problem, we develop a realistic model for the onset of the RNA world, using experimentally determined rates for polymerization reactions. We start with minimal assumptions about the initial state that only requires the presence of short oligomers or just free nucleotides and consider the effects of environmental cycling by dividing a day into a dry, semi-wet and wet phases that are distinguished by the nature of reactions they support. Long polymers, with maximum lengths sometimes exceeding 100 nucleotides, spontaneously emerge due to a combination of non-enzymatic, non-templated polymer extension and template-directed primer extension processes. The former helps in increasing the lengths of RNA strands, whereas the later helps in producing complementary copies of the strands. Strands also undergo hydrolysis in a structure-dependent manner that favour breaking of bonds connecting unpaired nucleotides. We identify the most favourable conditions needed for the emergence of ribozyme and tRNA-like structures and double stranded RNA molecules, classify all RNA strands on the basis of their secondary structures and determine their abundance in the population. Our results indicate that under suitable environmental conditions, non-enzymatic processes would have been sufficient to lead to the emergence of a variety of ribozyme-like molecules with complex secondary structures and potential catalytic functions.

Geochemical influences on nonenzymatic oligomerization of prebiotically relevant cyclic nucleotides.

The spontaneous emergence of long RNA molecules on the early Earth, a phenomenon central to the RNA World hypothesis, continues to remain an enigma in the field of origins of life. Few studies have looked at the nonenzymatic oligomerization of cyclic mononucleotides under neutral to alkaline conditions, albeit in fully dehydrated state. In this study, we systematically investigated the oligomerization of cyclic nucleotides under prebiotically relevant conditions, wherein starting reactants were subjected to repeated dehydration-rehydration (DH-RH) regimes. DH-RH conditions, a recurring geological theme that was prevalent on prebiotic Earth, are driven by naturally occurring processes including diurnal cycles and tidal pool activity. These conditions have been shown to facilitate uphill oligomerization reactions. The polymerization of 2'-3' and 3'-5' cyclic nucleotides of a purine (adenosine) and a pyrimidine (cytidine) was investigated. Additionally, the effect of amphiphiles was also evaluated. Furthermore, to discern the effect of "realistic" conditions on this process, the reactions were also performed using a hot spring water sample from a candidate early Earth environment. Our study showed that the oligomerization of cyclic nucleotides under DH-RH conditions resulted in intact informational oligomers. Amphiphiles increased the stability of both the starting monomers and the resultant oligomers in selected reactions. In the hot spring reactions, both the oligomerization of nucleotides and the back hydrolysis of the resultant oligomers were pronounced. Altogether, this study demonstrates how nonenzymatic oligomerization of cyclic nucleotides, under both laboratory-simulated prebiotic conditions and in a candidate early Earth environment, could have resulted in RNA oligomers of a putative RNA World.

Compositional heterogeneity confers selective advantage to model protocellular membranes during the origins of cellular life.

Protocellular membranes are thought to be composed of mixtures of single chain amphiphiles, such as fatty acids and their derivatives, moieties that would have been part of the complex prebiotic chemical landscape. The composition and physico-chemical properties of these prebiological membranes would have been significantly affected and regulated by their environment. In this study, pertinent properties were systematically characterized, under early Earth conditions. Two different fatty acids were mixed with their respective alcohol and/or glycerol monoester derivatives to generate combinations of binary and tertiary membrane systems. Their properties were then evaluated as a function of multiple factors including their stability under varying pH, varying Mg2+ ion concentrations, dilution regimes, and their permeability to calcein. Our results demonstrate how environmental constraints would have acted as important prebiotic selection pressures to shape the evolution of prebiological membranes. The study also illustrates that compositionally diverse membrane systems are more stable and robust to multiple selection pressures, thereby making them more suitable for supporting protocellular life.

NMR analysis of nucleotide π-stacking in prebiotically relevant crowded environment.

The prebiotic soup of a putative 'RNA World' would have been replete with a plethora of molecules resulting from complex chemical syntheses and exogeneous delivery. The presence of background molecules could lead to molecular crowding, potentially affecting the course of the reactions facilitated therein. Using NMR spectroscopy, we have analyzed the effect of crowding on the stacking ability of RNA monomers. Our findings corroborate that the purines stack more efficiently than the pyrimidine ribonucleotides. This competence is further enhanced in the presence of a crowding agent. This enhanced stacking could result in greater sequestration of the purine monomers, putting their ready availability for relevant nonenzymatic reactions into question. Thus, this study demonstrates the need for systematic characterization of molecular crowding in the context of prebiotically pertinent processes. Unraveling such phenomena is essential for our understanding of the transition from abiotic to biotic, during the origin of life.

Lipid-Assisted Polymerization of Nucleotides.

In addition to being one of the proponents of the "Lipid World hypothesis", David Deamer, together with other colleagues, pioneered studies involving formation of RNA-like oligomers from their 'non-activated', prebiotically plausible monomeric moieties. In particular, the pioneering work in this regard was a publication from 2008 in Origins of Life and Evolution of Biospheres, The Journal of the International Astrobiology Society, wherein we described the formation of RNA-like oligomers from nucleoside 5'-monophosphates. In that study, we had simulated a terrestrial geothermal environment, a niche that is thought to have facilitated the prebiotic non-enzymatic synthesis of polynucleotides. We showed that a mixture of lipids and non-activated mononucleotides resulted in the formation of relatively long strands of RNA-like polymers when subjected to repeated cycles of dehydration and rehydration (DH-RH). Since 2008, terrestrial geothermal niches and DH-RH conditions have been explored in the context of several other prebiotic processes. In this article, we review the work that we and other researchers have carried out since then in this line of research, including the development of new apparatus to carry out the simulation of prebiotic terrestrial geothermal environments.

Interaction of the mononucleotide UMP with a fluid phospholipid bilayer.

Interaction between mononucleotides and lipid membranes is believed to have played an important role in the origin of life on Earth. Studies on mononucleotide-lipid systems hitherto have focused on the influence of the lipid environment on the organization of the mononucleotide molecules, and the effect of the latter on the confining medium has not been investigated in detail. We have probed the interaction of the mononucleotide, uridine 5'-monophosphate (UMP), and its disodium salt (UMPDSS) with fluid dimyristoylphosphatidylcholine (DMPC) membranes, using small-angle X-ray scattering (SAXS), cryogenic scanning electron microscopy (cryo-SEM) and computer simulations. UMP adsorbs and charges the lipid membrane, resulting in the formation of unilamellar vesicles in dilute solutions. Adsorption of UMP reduces the bilayer thickness of DMPC. UMPDSS has a much weaker effect on interbilayer interactions. These observations are in very good agreement with the results of an all-atom molecular dynamics simulation of these systems. In the presence of counterions, such as Na+, UMP forms small aggregates in water, which bind to the bilayer without significantly perturbing it. The phosphate moiety in the lipid headgroup is found to bind to the hydrogens from the sugar ring of UMP, while the choline group tends to bind to the two oxygens from the nucleotide base. These studies provide important insights into lipid-nucleotide interactions and the effect of the nucleotide on lipid membranes.

Formation of Abasic Oligomers in Nonenzymatic Polymerization of Canonical Nucleotides.

Polymerization of nucleotides under prebiotically plausible conditions has been a focus of several origins of life studies. Non-activated nucleotides have been shown to undergo polymerization under geothermal conditions when subjected to dry-wet cycles. They do so by a mechanism similar to acid-catalyzed ester-bond formation. However, one study showed that the low pH of these reactions resulted in predominantly depurination, thereby resulting in the formation of abasic sites in the oligomers. In this study, we aimed to systematically characterize the nature of the oligomers that resulted in reactions that involved one or more of the canonical ribonucleotides. All the reactions analyzed showed the presence of abasic oligomers, with purine nucleotides being affected the most due to deglycosylation. Even in the reactions that contained nucleotide mixtures, the presence of abasic oligomers was detected, which suggested that information transfer would be severely hampered due to losing the capacity to base pair via H-bonds. Importantly, the stability of the N-glycosidic linkage, under conditions used for dry-wet cycling, was also determined. Results from this study further strengthen the hypothesis that chemical evolution in a pre-RNA World would have been vital for the evolution of informational molecules of an RNA World. This is evident in the high degree of instability displayed by N-glycosidic bonds of canonical purine ribonucleotides under the same geothermal conditions that otherwise readily favors polymerization. Significantly, the resultant product characterization in the reactions concerned underscores the difficulty associated with analyzing complex prebiotically relevant reactions due to inherent limitation of current analytical methods.

Templated replication (or lack thereof) under prebiotically pertinent conditions.

Accurate replication of encoded information would have been crucial for the formation and propagation of functional ribozymes during the early evolution of life. Studies aimed at understanding prebiotically pertinent nonenzymatic reactions have predominantly used activated nucleotides. However, the existence of concentrated pools of activated monomers on prebiotic Earth is debatable. In this study, we explored the feasibility of nonenzymatic copying reactions using the more prebiotically relevant 5'-nucleoside monophosphates (5'-NMP). These reactions, involving a 20-mer primer, were performed in the presence of amphiphiles, under volcanic geothermal conditions. Interestingly, the extended primer was not comparable to the expected full length 21-mer product. Our results suggest loss of the nitrogenous base in the extended primer. This phenomenon persisted even after lowering the temperature and when different rehydration solutions were used. We envisage that the loss of the informational moiety on the incoming 5'-NMP, might be occurring during addition of this monomer to the pre-existing oligomer. Significantly, when 5'-ribose monophosphate was used, multiple additions to the aforementioned primer were observed that resulted in hybrid polymers. Such hybrid oligomers could have been important for exploring a vast chemical space of plausible alternate nucleobases, thus having important implications for the origin of primitive informational polymers.

Correction: Synthesis of barbituric acid containing nucleotides and their implications for the origin of primitive informational polymers.

Correction for 'Synthesis of barbituric acid containing nucleotides and their implications for the origin of primitive informational polymers' by Chaitanya V. Mungi et al., Phys. Chem. Chem. Phys., 2016, 18, 20144-20152.