Incorporation of Basic α-Hydroxy Acid Residues into Primitive Polyester Microdroplets for RNA Segregation.

Nucleic acid segregation and compartmentalization were likely essential functions that primitive compartment systems resolved during evolution. Recently, polyester microdroplets generated from dehydration synthesis of various α-hydroxy acids (αHA) were suggested as potential primitive compartments. Some of these droplets can differentially segregate and compartmentalize organic dyes, proteins, and nucleic acids. However, the previously studied polyester microdroplets included limited αHA chemical diversity, which may not reflect the chemical diversity available in the primitive Earth environment. Here, we increased the chemical diversity of polyester microdroplet systems by combinatorially adding an αHA monomer with a basic side chain, 4-amino-2-hydroxybutyric acid (4a2h), which was incorporated with different ratios of other αHAs containing uncharged side chains to form combinatorial heteropolyesters via dehydration synthesis. Incorporation of 4a2h in the polymers resulted in the assembly of some polyester microdroplets able to segregate fluorescent RNA or potentially acquire intrinsic fluorescent character, suggesting that minor modifications of polyester composition can significantly impact the functional properties of primitive compartments. This study suggests one process by which primitive chemical systems can increase diversity of compartment "phenotype" through simple modifications in their chemical composition.

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.

Effect of Co-solutes on Template-Directed Nonenzymatic Replication of Nucleic Acids.

The widely acknowledged 'RNA world' theory pertains to how life might have chemically originated on early Earth. It presumes the existence of catalytic RNAs, which were also capable of storing and propagating genetic information. Substantial research has gone into understanding how enzyme-free reactions of nucleic acids might have led to the formation of such catalytic RNA polymers. However, most of these studies involved reactions that were performed in aqueous systems devoid of any "background" molecules. This scenario is not a true representation of the complex chemical environment that might have been prevalent on prebiotic Earth. In the present study, we analyzed the effect of co-solutes ("background" molecules) on the rate and accuracy of template-directed nonenzymatic replication of RNA, in a putative RNA world. Our results suggest that presence of co-solutes in the reaction affects the addition of purine monomers across their cognate template base. Reduction in the rate of these 'fast' cognate addition reactions resulted in an apparent increase in the frequency of mismatches in the presence of co-solutes. However, reactions that involved the addition of a mismatched base were not notably affected. Such a scenario could have led to an accrual of mutations during the propagation of functional sequences on early Earth, unless the relevant sequences were separated from the bulk reaction milieu by some limiting boundary structure (e.g., a membrane). In general, our results suggest that the presence of co-solutes could have affected certain prebiotic reaction rates to a larger extent than others. Even modest changes in nonenzymatic replication reaction rates could have eventually resulted in the accumulation of greater variation in RNA sequences over prolonged time periods. It, therefore, is pertinent to account for the chemical complexity intrinsic to prebiotic environments while studying relevant nonenzymatic reactions.

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.

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.

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.