Water cycles in a Hadean CO₂ atmosphere drive the evolution of long DNA

The study addresses how autonomous replication, mutation, and selection of genetic polymers could have proceeded under plausible Hadean Earth conditions without exposing RNA/DNA to damaging high temperatures. Classical thermal denaturation can exceed 100 °C for RNA in aqueous solution and promotes hydrolysis, while replication chemistries typically require elevated ion concentrations (for example, Mg²⁺), which stabilize duplexes and hinder strand separation. Alternative denaturation mechanisms (ionic oscillations, low pH, chaotropes) lack clear integration into an autonomous replicator. A further challenge is Spiegelman’s ‘tyranny of the shortest’, whereby shorter strands outcompete longer ones, eroding genetic information. The authors hypothesize that cyclic dew formation in a CO₂-rich Hadean atmosphere could create alternating microenvironments—acidic, low-salt dew and neutral, salt-rich bulk—enabling low-temperature strand separation and reannealing/replication, while interfacial transport concentrates and selects longer strands. They propose that such cycles drive sequence evolution, overcoming the bias toward short oligomers and allowing emergence of long, AT-biased sequences adapted to the local melting landscape.

Background work highlights constraints on prebiotic replication: salt- and temperature-dependent stability of nucleic acid duplexes; necessity of strand separation for replication; deleterious effects of high-temperature denaturation on RNA integrity; and prior proposals for alternative denaturation (low pH, chaotropes, ionic oscillations). Spiegelman’s experiments established kinetic dominance of short replicators, framing the selection problem. Geological literature suggests high Hadean pCO₂ (0.1–10 bar), implying acidic waters upon dissolution (carbonic acid/bicarbonate systems) and low pH in condensed, low-ionic-strength water such as dew. Prior experimental/theoretical studies from the authors and others showed non-equilibrium accumulation of nucleotides/oligonucleotides in thermal gradients, coffee-ring effects, and wet–dry cycling promoting condensation/replication processes. These works motivate testing CO₂-driven dew cycles as an integrated, autonomous denaturation–replication–selection mechanism.

Experimental system: A microfluidic dew chamber (250 µm × 30 mm × 14 mm) composed of Teflon, a sapphire window (cold side), and a copper back plate over a silicon wafer (hot side) generated controlled temperature gradients (5–17 °C) with precise control (±1 °C). Approximately 20 µl of solution (∼1/3 volume) was introduced, leaving 2/3 gas volume. The gas phase was enriched with CO₂ (0.1–1 bar pCO₂). Evaporation at the warm side and condensation at the cold side produced dew droplets that grew, fused, and intermittently formed capillary bridges; droplets were initially salt-free, absorbing CO₂ to reach low pH, while the bulk remained salt-rich and near-neutral pH. Measurements: pH mapping used Lysosensor Yellow/Blue DND-160 (20 µM) and a custom fluorescence microscope with multi-LED excitation and ratiometric detection. RNA duplex fraction was assessed via FRET with centrally labeled complementary 24-bp RNA duplexes of defined GC content (e.g., 33% GC; Tm 66 °C) in Tris 10 mM with MgCl₂ 10–12.5 mM, initial pH 7.0. FRET imaging was conducted under gradients (e.g., 31–43 °C; average ∼29 °C below Tm). Dew and bulk were analyzed separately. Modeling: A pH model based on CO₂ absorption kinetics into pure water and buffered solutions predicted dew vs bulk pH across pCO₂. Hybridization thermodynamics (Van ’t Hoff-based) were parameterized from melting curves across pH, Mg²⁺, and sequences to compute duplex fraction (RNA/DNA FRET) as a function of conditions; fitted dew Mg²⁺ was 0.1–0.5 mM (∼50–100× reduction vs bulk). Transport simulations used 3D finite-element COMSOL models to capture convection, capillary flow, diffusion, thermophoresis, and interfacial geometries matching experiments under a 17 °C gradient (hot 60 °C, cold 43 °C). DNA diffusion coefficients scaled with length; steady-state accumulation factors at gas–liquid interfaces were computed for 1–300 nt. Replication experiments: To emulate prebiotic replication dynamics, enzymatic DNA polymerization (QIAGEN Taq PCR Master Mix 1×: MgCl₂ 1.5 mM in final mix; Tris ∼10 mM; dNTPs 200 µM each; Taq 2.5 U µl⁻¹; SYBR Green I 2×; BSA 0.1%) was performed within the dew chamber following a 95 °C, 3 min activation. Temperature gradients included 67/52 °C (Δ15 °C) for a 51-bp template (Tm 88 °C) and 60/43 °C (Δ17 °C) for competitive templates (47 nt, Tm 78 °C; 77 nt, Tm 89 °C). Reactions proceeded for 5–12 h with 1 bar pCO₂; controls at ambient CO₂ were run. Products were visualized by SYBR fluorescence and analyzed by denaturing PAGE (12.5–15% acrylamide, 50% urea) with SYBR Gold staining. Sequencing and analysis: Products from CO₂-dew replication were prepared for Oxford Nanopore MinION sequencing (end-repair/A-tailing, barcoding, adapter ligation, bead purifications) and sequenced for 24 h (Guppy v3.6 basecalling), yielding 2,856 reads (140–1,300 nt). Sequence composition (AT:GC), k-mer (4-mer) frequencies, and Shannon entropy relative to random pools were computed. A stochastic replication model simulated unspecific templation: sequence replication probability weighted by the dew DNA FRET (stability) landscape; each replication generated a reverse complement plus a random insertion/deletion of a random subsequence and random point mutations at Taq-like error rates (10⁻⁴). Parameter sweeps tested temperature effects on the AT:GC outcomes.

• Dew–bulk microenvironments form under Hadean-like CO₂: at pCO₂ = 1 bar, dew droplets acidify to pH ∼4.0 while bulk remains buffered (pH 5.8 ± 0.2 with Tris), matching model predictions.
• Low-salt, low-pH dew melts RNA at low temperature: dsRNA FRET in dew dropped from 0.78 ± 0.05 (ambient pCO₂) to 0.17 ± 0.08 (pCO₂ ≥ 0.8 bar) under 31–43 °C; bulk remained nearly fully duplexed. Effective dew Mg²⁺ 0.1–0.5 mM (∼50–100× lower than bulk), enabling melting ∼30 K below bulk Tm without high-temperature spikes.
• Strong interfacial accumulation and length selection: COMSOL and fluorescence show continuous coffee-ring-driven accumulation at gas–water interfaces. Accumulation factors reached >10,000-fold for 77-nt DNA; length dependence favored longer strands due to reduced diffusivity, up to >20,000-fold for 300-nt DNA at the bulk interface (∼1,200-fold at dew interface).
• DNA replication at temperatures far below Tm: A 51-bp template (Tm 88 °C) amplified in the dew chamber with an average temperature 28 °C below Tm, showing exponential SYBR signal increase; no replication at ambient CO₂ (insufficient melting).
• Overcoming ‘tyranny of the shortest’: In standard thermocycling, 47-nt outcompeted 77-nt templates; in CO₂-dew cycles (60/43 °C, 1 bar pCO₂, ∼12 h), both templates replicated comparably and additional, longer DNAs appeared, consistent with interfacial enrichment and wet–dry-driven recombination/templation.
• Emergence of long, AT-rich sequences: Nanopore reads spanned 140–1,300 nt; longer reads exhibited AT fractions >80%, with loss of simple primer/template repeats at >300 nt. Sequence compositions mapped to regions of low duplex fraction (DNA FRET ≈ 0.14 ± 0.12) under dew conditions, indicating selection for sequences optimally cycling between denaturation and templating.
• Convergent experiment–model fingerprints: 4-mer analysis showed enrichment of AT-rich motifs (e.g., ATAT, AATA; model also AAAA, TTAT) versus random. Informational entropy reduced by 44% (experiment; minimum relative entropy 0.56) and 34% (model; 0.66), indicating selection into a constrained sequence subspace.
• Environmental control of composition: Increasing average chamber temperature (fixed ΔT = 17 °C) shifted replicated pool from AT-rich to GC-richer: experiment AT:GC decreased from 4.7 ± 1.1 (51 °C) to 0.9 ± 0.6 (67 °C); model from 8.0 ± 1.0 (50 °C) to 0.9 ± 0.1 (75 °C). Similar evolutionary trajectories observed starting from GC-rich short templates in simulations.

The findings demonstrate that CO₂-driven dew cycles provide an autonomous mechanism to couple low-temperature strand separation with reannealing/replication under salt-rich bulk conditions, addressing a central bottleneck for prebiotic replication. The synergy of low pH and low ionic strength in dew enables denaturation ∼30 K below bulk Tm, minimizing hydrolysis, while the buffered bulk supports annealing and enzymatic (proxy) replication. Non-equilibrium interfacial flows concentrate nucleic acids and selectively enrich longer strands, counteracting kinetic advantages of short oligomers and overcoming Spiegelman’s ‘tyranny of the shortest’. The evolving DNA pool adapts to the dew’s stability landscape: sequences with intermediate stability (optimized for repeated melt–template cycles) are preferentially replicated, driving AT enrichment and repetitive motifs and reducing sequence entropy to a manageable subspace where functional sequences could emerge. Temperature-tunable selection demonstrates environmental control over sequence composition, consistent with the hypothesis that early Earth microenvironments could steer molecular evolution. Together, the results support a plausible physical–chemical route for early Darwinian evolution of long nucleic acids in heated rock pores under Hadean-like atmospheres.

A CO₂-rich dew cycle in a heated rock-pore analogue supplies key features for a prebiotic molecular replicator: cyclic, low-temperature denaturation; salt-rich reannealing conditions; strong interfacial concentration; and length-selective accumulation that promotes the survival and replication of longer strands. These out-of-equilibrium cycles drive the emergence of long, AT-biased DNA sequences with reduced informational entropy, effectively navigating an otherwise vast sequence space and overcoming the dominance of short oligomers. The platform offers a physically plausible setting for early molecular evolution and suggests that environmental parameters (temperature, pCO₂, Mg²⁺, pH) could have steered nucleotide composition on the early Earth. Future work should replace enzymatic polymerases with prebiotically plausible non-enzymatic chemistries or ribozymes, explore RNA-focused replication and stability, assess mineral-buffer interactions over longer timescales, and test robustness across varied geochemical conditions and gas compositions relevant to Hadean environments.

The replication chemistry employed a modern enzyme (Taq polymerase) as a proxy for prebiotic processes; direct demonstration of non-enzymatic or ribozyme-mediated replication under dew cycles remains to be shown. pH imaging and FRET measurements were conducted at temperatures differing from some replication runs, requiring model-based extrapolation of pH behavior; imaging dyes and conditions impose technical constraints. The laboratory microfluidic pore simplifies natural heterogeneities (mineralogy, porosity, multiscale flows), and long-term mineral buffering effects occur over days, beyond the timescale of seconds–hours dew cycles. Nanopore sequencing has characteristic error profiles and length detection limits (reads <140 nt not captured), potentially biasing composition analysis. The system focuses on DNA proxies; RNA stability and replication kinetics under identical conditions need thorough quantification. Absolute interfacial concentration factors in nature may vary with pore geometry and environmental fluctuations.