Emergent ribozyme behaviors in oxychlorine brines indicate a unique niche for molecular evolution on Mars

The possibility of life on Mars has long attracted interest, particularly due to early Noachian-era conditions that were more Earth-like and conducive to prebiotic chemistry. Evidence for wet-dry cycling, stabilizing borates, and phyllosilicate clays suggests plausibility for prebiotic reactions such as sugar stabilization and RNA polymerization. However, present-day Mars lacks bulk liquid water and hosts widespread oxychlorine species (notably perchlorate), which are toxic and reactive yet capable of forming deliquescent brines that may persist near or below the surface and be shielded from ultraviolet radiation by shallow regolith. Against this geochemical backdrop, the authors hypothesize that RNA-based catalysts (ribozymes) may be intrinsically resilient to perchlorate-rich hypersaline conditions relative to protein enzymes, and that oxychlorine chemistry could even enable emergent ribozyme functions (e.g., regulatory behaviors and halogenation chemistry). The study tests how perchlorate and related oxychlorine species affect the folding, activity, reversibility of denaturation, and potential new functions of representative ribozymes and proteins, to assess whether Martian brines could constitute a niche for RNA-driven molecular evolution.

Prior work indicates early Mars had conditions favorable to prebiotic chemistry, including wet-dry cycles that concentrate reactants and promote dehydration-condensation reactions, borates that stabilize sugars (evidence from ChemCam at Gale Crater), and clays that can catalyze RNA oligomerization (identified by MRO). Multiple missions (Phoenix, Curiosity) and meteorite analyses have shown that oxychlorine species (perchlorate, chlorate, chlorite) are widespread on Mars, possibly forming via surface-atmosphere processes and photochemistry, and present since at least the Noachian-Hesperian. Perchlorate’s hygroscopicity suggests near-surface brines could persist, while UV radiation hazards can be mitigated beneath millimeters of regolith. Terrestrial halophilic organisms adjust protein surface charge for salt tolerance, but proteins often denature in high salt, especially chaotropic salts like perchlorate. In contrast, nucleic acids, being polyanionic, often retain structure/function in high salt and even in nonaqueous ionic media. These observations motivate testing whether functional RNAs outperform proteins under Martian-relevant oxychlorine brine conditions and whether oxychlorine species could support novel ribozyme chemistries.

The study compared functional RNAs and proteins under varying chloride and oxychlorine salt concentrations, focusing on perchlorate. Assays included: - Hammerhead ribozyme self-cleavage kinetics: 100 nM each of hammerhead strands A and B in buffers containing 50 mM Tris-HCl pH 8 and 2.5 mM Mg2+, with NaClO4 (and NaCl controls) spanning up to ~6 M; reactions initiated with Mg2+, quenched by ethanol, products resolved on 20% urea-PAGE, quantified via 5′-fluorescein labeling and GelQuant. - Protein restriction nuclease assays: EcoRI, NheI, and TaqI-v2 tested in manufacturer buffers (EcoRI buffer or rCutSmart) with duplex DNA substrates. EcoRI incubated at 37 °C; TaqI-v2 at 65 °C. Activity assessed by gel electrophoresis and fluorescence labeling; quantified with GelQuant. - Broccoli aptamer fluorescence and melting: 2 µM Broccoli RNA with 50 µM DFHBI-1T in 50 mM HEPES pH 7.4, 5% DMSO, 100 mM MgX (X = Cl− or ClO4−), and variable NaX to achieve target salt concentrations up to near saturation. Fluorescence melting profiles collected on a BioRad CFX96 to assess cooperative folding. - RNA polymerase ribozyme (C19Z) primer extension: 500 nM C19Z, 500 nM RNA template and primer (primer 5′-fluorescein labeled), 4 mM NTPs, 50 mM Tris-HCl pH 8.3, 200 mM MgX, with NaX adjusted for chloride/perchlorate concentration. Anneal at 80 °C then chill; reactions started by adding primer and salt, incubated at 4 °C for 20 min, products ethanol-precipitated, displaced with DNA complement, resolved by 20% urea-PAGE, quantified with GelQuant. - β-lactamase (HaBlα) nitrocefin hydrolysis: 5 µM enzyme with 50 µM nitrocefin in 5 mM phosphate pH 7; perchlorate varied. Absorbance at 486 nm measured; extinction coefficient corrections applied for high-salt conditions via fully hydrolyzed nitrocefin standards in matched brines. - Perchlorate recovery assays: RNAs or proteins pre-incubated 30 min at 4 °C in high perchlorate (generally up to 5 M), then diluted into lower-salt assay conditions; activity compared to positive and negative controls to assess reversibility of denaturation. - Chlorination (halogenation) assays with oxychlorine species: G-quadruplex RNA–hemin holoenzyme (e.g., rPS2M/HemE) or HRP (control) catalyzed chlorination of monochlorodimedone (MCD, 50 µM) or phenol red (50 µM) in 100 mM Li-HEPES pH 7.4 with hemin (5 µM), RNA (10 µM), and NaClO2 or NaClO4 as oxidant; kinetics monitored by UV–Vis (e.g., A250 for MCD, A574 for phenol red). Products analyzed by ESI-MS after C18 extraction to identify chlorinated species. - G-quadruplex/hemin peroxidase-like redox assay: RNA heat-cycled with NaClO3, complexed with hemin, then reacted with Amplex Red and H2O2; fluorescence measured to confirm catalytic redox activity across salt conditions. All gels imaged on an Omega Lum system; spectroscopic data acquired on standard plate readers or UV–Vis spectrophotometers; data processed in Igor Pro 9.0.

- Hammerhead ribozyme activity exhibited a biphasic response with a rate maximum at ~5 M NaClO4 (kapp ≈ 0.79 h−1) and retained catalysis up to 6 M NaClO4 (kapp ~ 0.24 h−1). In 5 M NaCl, cleavage was faster (1.48 h−1), ~1.9× higher than in 5 M NaClO4. - The mesophilic restriction enzyme EcoRI lost function at much lower perchlorate (as low as 0.2–0.5 M NaClO4). Where active, EcoRI showed ~10-fold slower kinetics in perchlorate versus chloride (kapp ≈ 0.04 h−1 in NaClO4 vs 0.48 h−1 in NaCl). - The Broccoli aptamer retained ligand-activated fluorescence and cooperative folding up to 5 M NaClO4, but fluorescence decreased substantially with perchlorate: ~80% reduction at 2 M and ~94% reduction at 5 M relative to chloride controls. - The RNA polymerase ribozyme C19Z showed gradual loss of yield with increasing perchlorate (Δyield/Δ[ClO4−] ≈ −0.74% per M), retained extension activity up to 3.5 M perchlorate (including extension to ~37 nt), and lost activity at 4 M. Yields in perchlorate were a little over half of those in chloride (chloride slope ~ −1.19% per M). - Extremophilic protein TaqI-v2 (from Thermus aquaticus) nevertheless was highly perchlorate-sensitive, losing activity by ~0.2 M NaClO4, despite retaining activity in 0.2 M NaCl (kapp ≈ 0.21 h−1). - Recovery from perchlorate-induced denaturation: RNAs (hammerhead, Broccoli, C19Z) uniformly recovered activity after dilution from high to low salt. Among proteins, TaqI-v2 regained activity upon dilution, HaBlα recovered partially (~30%), and EcoRI was irreversibly inactivated with no recovery. - Perchlorate and other oxychlorine species enabled emergent ribozyme functions: a G-quadruplex RNA–hemin holoenzyme (rPS2M/HemE) catalyzed chlorination reactions (e.g., of monochlorodimedone and phenol red) using chlorite as oxidant/electron acceptor, with kinetics observable by UV–Vis and products verified by ESI-MS (chlorinated phenol red species detected). Comparative assays with HRP established analogous catalytic capability by the RNA–heme complex. - Overall, functional RNAs demonstrated tolerance to perchlorate concentrations at least an order of magnitude higher than mesophilic proteins, maintained fold-dependent activity in hypersaline oxychlorine brines, and displayed reversible denaturation behavior, whereas proteins were generally more susceptible and less recoverable.

The results directly support the hypothesis that RNAs are intrinsically more compatible with perchlorate-rich hypersaline brines than protein enzymes. The hammerhead ribozyme, Broccoli aptamer, and C19Z polymerase retained function at multi-molar perchlorate concentrations, in some cases near NaClO4 saturation, and recovered activity upon dilution after high-salt exposure. In contrast, mesophilic proteins (e.g., EcoRI) lost activity at submolar perchlorate and often failed to recover, while even an extremophilic enzyme (TaqI-v2) showed high perchlorate sensitivity. These outcomes are consistent with a model wherein the polyanionic nucleic acid backbone resists salt-induced hydrophobic disruption that often destabilizes protein folding. Beyond tolerance, oxychlorine species enabled emergent ribozyme functions, including catalytic halogenation via a G-quadruplex–hemin RNA holoenzyme, demonstrating that Martian-relevant oxidants can be harnessed by RNA-based catalysts to perform specific chemical transformations. Collectively, these findings imply that near- or subsurface oxychlorine brines on Mars could constitute a favorable niche for RNA-mediated chemistry and molecular evolution, potentially facilitating homeostasis-like regulatory behaviors and novel reactivities under conditions inhospitable to many proteins.

This work demonstrates that functional RNAs maintain folding, catalysis, and reversible denaturation in highly concentrated perchlorate brines, while representative proteins are strongly inhibited or irreversibly denatured. Oxychlorine species can further enable emergent ribozyme functions such as catalytic halogenation when complexed with hemin in G-quadruplex scaffolds. Together, these results position nucleic acids as uniquely well-suited to hypersaline Martian environments and suggest that oxychlorine brines could have provided a distinctive niche for RNA-centered molecular evolution on Mars. Future research should examine broader classes of ribozymes and protein enzymes, assess additional Martian-relevant salts (e.g., mixed perchlorates, magnesium/calcium salts), temperature and wet–dry cycling effects, radiation and regolith interactions, and the potential for coupled regulatory networks and metabolism-like pathways driven by RNA in realistic brine chemistries.

Experiments were conducted in vitro under controlled laboratory conditions (defined buffers, temperatures, and salt compositions) that may not capture the full complexity of Martian environments (e.g., temperature extremes, radiation flux, mixed salt mineralogy, wet–dry cycling dynamics). The study tested a limited set of RNAs and proteins, so generalizability across all biopolymers is uncertain. Perchlorate was primarily studied as sodium salts with specified Mg2+ levels and specific pH conditions, which may differ from Martian brine compositions and physicochemical parameters. While emergent halogenation activity was demonstrated for a G-quadruplex–hemin RNA under certain oxychlorine conditions, the breadth of such chemistries across substrates and oxidants and their long-term stability in Martian-like settings remains to be determined.