The possibility of past or present life on Mars is a compelling question in astrobiology. Similarities between early Earth and Mars during the Noachian period, characterized by liquid water and reducing environments conducive to prebiotic chemistry, suggest independent life origins may have occurred on both planets. Evidence for wet-dry cycling during the Noachian-Hesperian transition further supports this, as these cycles concentrate reactants and promote reactions like the formation of nucleosides and RNA polymerization. Chemical species like borates (stabilizing sugars) and phyllosilicate clays (promoting RNA polymerization) have been identified on Mars, adding weight to the possibility of prebiotic chemistry. However, challenges to present-day life exist, primarily the scarcity of liquid water due to low temperatures and atmospheric pressure, resulting in the desiccation of Noachian oceans. Another significant obstacle is the abundance of perchlorate (ClO₄⁻) and other oxychlorine species across Mars' surface, detected by various missions. These oxychlorine compounds are toxic to terrestrial life and their toxicity is exacerbated by UV radiation. However, perchlorate salts are hygroscopic, suggesting near-surface brines could act as a refuge for extant life. Furthermore, even limited regolith thickness can significantly reduce UV radiation, suggesting the subsurface may be hospitable. These brines, while providing liquid water, introduce a new challenge: perchlorate is reactive and destabilizing to many biomolecules. This research examines the impact of perchlorate brines on microbial evolution on Mars, contrasting moderately saline environments like the Noachian oceans with hypersaline brines in present-day Martian conditions. We hypothesize that if Martian life emerged, it may have followed evolutionary paths different from those on Earth, potentially leading to perchlorate tolerance in ribozymes, a class of RNA molecules with catalytic properties.

Extensive research supports the possibility of past habitable conditions on Mars, particularly during the Noachian era. Studies have focused on the presence of liquid water, mineral composition, and the potential for prebiotic chemical reactions. The discovery of borates and clays, both essential for prebiotic processes, further strengthens this possibility. However, the challenges posed by present-day Mars conditions, including low water activity and the presence of toxic perchlorates, have also been investigated. The research community has proposed various mechanisms for perchlorate formation, both abiotic and potentially biotic. The effects of perchlorates on terrestrial life and the potential for adaptation are also areas of ongoing research. Understanding the interactions between perchlorates and potential biomolecules, specifically in the context of Martian hypersaline environments, is crucial for evaluating the habitability of Mars. Several models of potential Martian brines and their implications for microbial life have been studied. The role of recurring slope lineae (RSLs) as potential sites for transient wet-dry cycles and the presence of subglacial reservoirs are significant considerations.

The study investigated the effects of perchlorate and other oxychlorine species on the function of both ribozymes (RNA enzymes) and protein enzymes. Several ribozymes were tested, including the hammerhead ribozyme (a self-cleaving RNA), the Broccoli aptamer (an RNA that binds a specific ligand), and the C19z ribozyme polymerase (an RNA that catalyzes RNA synthesis). The protein enzymes tested included EcoRI (a restriction enzyme), TaqI (a thermostable restriction enzyme from a thermophilic organism), and RNase H1. The experiments involved measuring the activity of these biomolecules in solutions containing varying concentrations of perchlorate and other salts (NaCl, MgCl2). The effect of perchlorate on ribozyme activity was assessed by measuring the rate of self-cleavage (hammerhead), ligand binding (Broccoli), and RNA polymerization (C19z). Protein enzyme activity was assessed by measuring the rate of DNA cleavage (EcoRI, TaqI) or hydrolysis of nitrocefin (β-Lactamase). The reversibility of perchlorate-induced denaturation was tested through dilution assays. Additionally, the study examined the impact of other oxychlorine species (chlorite, hypochlorite) on ribozyme function and their potential role in ribozyme-catalyzed chlorination reactions. Kinetic assays and mass spectrometry were used to characterize these reactions. The experimental setup for each assay varied slightly to accommodate the specific properties of each ribozyme and protein. For instance, the hammerhead ribozyme assay involved monitoring self-cleavage using gel electrophoresis, while the Broccoli aptamer assay measured fluorescence. The methods used for data analysis included gel electrophoresis quantification, spectrophotometry, and fluorescence measurements. The data was analyzed using Igor Pro 9.0.

The study's key findings include: 1. Functional RNAs (ribozymes) exhibited significantly greater tolerance to perchlorate than mesophilic protein enzymes. Specifically, ribozymes retained function at perchlorate concentrations (3-6 M) at least 10-fold higher than those that inactivated the protein enzymes tested. The hammerhead ribozyme showed a biphasic response, with activity increasing to a maximum at 5 M NaClO4 before decreasing. The Broccoli aptamer also demonstrated high perchlorate tolerance. The C19z ribozyme polymerase showed reduced activity at higher perchlorate concentrations but retained some function even at 3.5 M NaClO4. 2. Mesophilic RNAs showed a capacity to recover function after perchlorate-induced denaturation, a property more commonly observed in extremophilic proteins. In contrast, the mesophilic proteins EcoRI and RNase H1 showed irreversible inactivation at lower perchlorate concentrations. The extremophile TaqI-v2 exhibited some recoverability. 3. Perchlorate enabled emergent ribozyme functions, such as acting as an electron donor/acceptor in chlorination reactions. A ribozyme showed the ability to catalyze chlorination reactions using chlorite as a reactant. This suggests that perchlorate may have influenced the evolution of ribozymes on early Mars by enabling new regulatory functions. The results indicate that RNA is uniquely suited to survive and function in the hypersaline oxychlorine brines thought to exist on Mars, suggesting these brines could have been a suitable niche for molecular evolution. The observed ribozyme resilience to perchlorate contrasts sharply with the sensitivity of mesophilic protein enzymes, supporting the idea that RNA might have played a more significant role in early Martian life than previously considered. The ability of ribozymes to recover from perchlorate-induced denaturation reinforces this notion of inherent RNA resilience in harsh environments.

This study's findings strongly suggest that RNA-based life might have been, or may still be, viable in Martian hypersaline environments. The remarkable tolerance of ribozymes to high perchlorate concentrations, along with their ability to recover functionality after denaturation, challenges previous assumptions about the limitations of life in such environments. The emergent catalytic functions facilitated by perchlorate further expands the potential for RNA-based life on Mars, demonstrating an unexpected adaptation to a previously considered hostile environment. The observed differences between RNA and protein responses to perchlorate highlight the importance of considering different biomolecular structures and their unique physicochemical properties when assessing potential extraterrestrial life. These findings expand our understanding of the potential for life beyond Earth, particularly in environments considered extreme by terrestrial standards. Further research should focus on exploring the potential for other unique adaptations of life in extreme hypersaline environments, especially in the context of prebiotic chemistry and early molecular evolution on Mars. Investigating the potential role of other oxychlorine species in influencing biomolecular evolution should also be prioritized.

This research demonstrates the remarkable tolerance and adaptability of ribozymes to hypersaline oxychlorine brines, conditions thought to exist on Mars. The unique emergent functions enabled by perchlorate suggest a potential niche for RNA-based life in this environment. These findings significantly expand our understanding of potential habitats for extraterrestrial life and the role of RNA in early molecular evolution. Future research could focus on more detailed investigations into the specific mechanisms underlying ribozyme tolerance to perchlorate, exploring the potential for other novel ribozyme functions in these brines, and searching for evidence of such adaptation in Martian samples.

While this study provides compelling evidence for ribozyme tolerance to perchlorate, further research is needed to confirm these findings in more complex Martian-simulated environments. The study focused on specific ribozymes and proteins, and the results may not generalize to all biomolecules. The use of laboratory-based simulations may not perfectly replicate all aspects of the Martian environment. Therefore, the results should be interpreted cautiously, and further in-situ investigations are necessary to validate these findings on Mars itself.