Selection of cycling conditions

When choosing conditions for model prebiotic experiments, it is necessary to balance historical plausibility with experimental convenience. Although an Earth day was shorter at 4 Ga30, we selected cycles that lasted 24 h as a reasonable approximation that was also convenient to monitor over many days. The hot-and-dry phases were set to 100 °C or 120 °C, temperatures in line with those found in previous studies31,32. While several previous studies relied on longer drying times33—which generally produce higher yields of condensation—long periods of heat (>6 h) seem unlikely given the 24-h diurnal cycle on Earth. We therefore limited our heating periods to 4–6 h per cycle.

For the cool phase of each cycle, the relative humidity (%RH) was varied from 30–70 %RH. Given the wide range of %RH values on modern Earth—from single digits in desert climates to 100% when raining—these values are both reasonable in a historical context and convenient, as they are easily simulated in the laboratory. Values of %RH much higher or lower are considerably more difficult for a humidity chamber to maintain with precision.

We maintained the temperature at 40 °C for the cool phase of the wet–dry cycles. A temperature slightly elevated from room temperature was necessary to ensure that a constant temperature, and relative humidity could be maintained despite daily fluctuations in the ambient conditions of the laboratory, e.g., a particularly warm or humid day. The higher cool temperature seems relevant to early Earth, as there is geological evidence it was generally warmer than today34.

Selection of model reaction and substrates

We targeted the oligomerization of amino acids into peptides as a model condensation reaction for our study for its obvious relevance to prebiotic chemistry. Glycine is the simplest and most prebiotically relevant amino acid. It is produced in the highest yields in simulated prebiotic syntheses of amino acids2,35,36,37, and a multifactor analysis by Trifonov suggests that glycine was the most prevalent amino acid in the earliest proteins38. As a matter of experimental rigor, yields of oligoglycines can be measured quantitatively by liquid chromatography14,39. Glycine is the only canonical amino acid that produces achiral oligomers, which simplifies their analysis relative to other amino acids. Chromatographic analysis is crucial because the high quantity of salts present greatly complicates quantitative analysis by mass spectrometry (see Supplementary Fig. 30, Supplementary Table 25, and related discussion in the Supplementary Discussion). The results from our experiments are summarized in Supplementary Tables 1 and 2.

Deliquescence regulates reversible and limited hydration

We first examined the hydration of a simple mixture of glycine with K+, Na+, Cl−, and OH− salts. Chloride salts are the water-soluble minerals most abundant on Earth40. Hydroxide was selected as a simple source of base to catalyze condensation, which will proceed less efficiently near neutral pH14. While neither KCl nor NaCl are deliquescent by themselves at 70 %RH, a mixture of the two and their hydroxides lowered the critical relative humidity (RH 0 ) to the point where the mixture was deliquescent. The reaction mixture used for the initial cycling experiments had a 20:10:1 molar ratio of chlorides:glycine:hydroxides with a 3:1 molar ratio of Na+:K+ (see Supplementary Methods and Supplementary Figs. 2 and 3).

Identical samples of 50 mg of the salt mixture in glass scintillation vials were cycled with cooling phases at 40 °C and three different relative humidities: 30, 50, and 70 %RH. The samples at 70 %RH—a value above the critical RH 0 of the mixture—collected and lost ~0.05 g of water per cycle, enough to create homogeneous aqueous solutions during the cool phases. The samples at 30 and 50 %RH—below the critical RH—varied by <0.01 g and remained white solids throughout both the hot and cool phases of ten complete cycles. Figure 1 shows the change in mass (corresponding to moisture loss and uptake) over time for the 30 and 70 %RH samples, demonstrating not only how deliquescence (dependent on the RH 0 value) can have a profound influence on the rehydration of identical samples, but also on the precise control of this rehydration in terms of reproducibility and reversibility.

Fig. 1 Demonstration of deliquescent wet–dry cycles. a An illustration of our deliquescent model with its cycling conditions. b A demonstration of the magnitude, reversibility, and consistency of moisture sorption/desorption during wet–dry cycling of a 50 mg mixture of K+, Na+, Cl−, OH–, and glycine. Containers were weighed over the course of ten complete 24-h cycles to monitor moisture gain and loss at 70 %RH (red triangles, above RH 0 for the mixture) and 30 %RH (blue circles, below RH 0 for the mixture). Each cycle consisted of two phases: a cool phase for 20 h at 40 °C (blue background) followed by a hot phase for 4 h at 120 °C (orange background). The error bars represent 95% confidence intervals (n = 3 identical experiments). Source data are provided as a Source Data file Full size image

Effects of humidity on yields of oligomerization

Next, we tested the ability of these mixtures of salts to host the condensation of glycine by wet–dry cycling with rehydration mediated by changes in atmospheric humidity. Samples were subjected to ten full 24-h cycles of heating (at 100 °C or 120 °C) and cooling (at 30, 50, or 70 %RH and 40 °C). For samples subjected to 100 °C, the heating phases lasted 6 h and the cooling phases lasted 18 h. For samples subjected to 120 °C, the heating phases lasted 4 h and the cooling phases lasted 20 h.

Figure 2 shows the total yield of glycine oligomers (Gly ≥2 ) for the six combinations of heating and cooling conditions. Regardless of the temperature of heating, the samples that were deliquescent and formed aqueous solutions during the cooling phase (at 70 %RH) produced substantially higher yields than those that were not deliquescent and remained solid throughout the experiment (at 30 and 50 %RH). All samples that did not rehydrate due to insufficient humidity for deliquescence produced only limited yields of oligomers, below 2.5%. At 70 %RH, where the mixtures were deliquescent, those subjected to heating at 120 °C produced higher total yields of oligomer after a lesser number of cycles, but by the end of the experiment (ten cycles), the samples heated at 100 °C were comparable. Supplementary Figs. 4–9 and Supplementary Tables 4–9 show histograms that track the yields of each oligomer measured at the end of cycles 1, 2, 3, 5, and 10. There is a clear advantage to periodic rehydration of these samples (at 70 %RH) versus remaining dry throughout (at 30 and 50 %RH).

Fig. 2 The condensation of glycine in a deliquescent system. a The condensation of glycine into oligoglycines. We report yields based on the percentage of initial glycine converted into oligomers, excluding cyclic DKP dimer. b The total yields of glycine oligomers in the presence of (K+/Na+)(−Cl/−OH) after 1, 2, 3, 5, and 10 cycles. Each cycle was 24 h. For the red samples, one cycle included 20 h at 40 °C and 70 %RH (circles), 50 %RH (diamonds), or 30 %RH (triangles) followed by 4 h at 120 °C. For the blue samples, one cycle included 18 h at 40 °C and 70, 50 or 30 %RH (circles, diamonds, triangles) followed by 6 h at 100 °C. The error bars represent 95% confidence intervals (n = 3 identical experiments). The downturn in total yield observed at higher temperature is addressed in the Supplementary Discussion. Source data are provided as a Source Data file Full size image

In a control experiment, we tested glycine oligomerization starting from homogeneous solutions of the chloride mixtures as opposed to dry mixtures. In these experiments, all of the sample mixtures started as wet in the first cycle before being subjected to cycling with either 30 or 70 %RH cool periods. We observed that both sets of mixtures gave comparable yields at the end of the first cycle, but the samples cooled at 70 %RH (such that every cycle had both a wet and a dry phase) gave considerably higher yields for all subsequent cycles versus those cooled at 30 %RH, which dried after the first cycle and remained dry because RH 0 was never surpassed (see Supplementary Figs. 12–19 and Supplementary Tables 12–17).

Benefits of regulated rehydration

The results above are consistent with the idea that the increased diffusion of reactants permitted in solution more than compensates for hydrolysis that occurs in the presence of the extra water17,18. But there is clearly a limit to the benefits of water in these systems. In an experiment designed to study the effects of overhydration, the same mixtures of glycine with K+, Na+, Cl−, and OH− salts were subjected to phases of 6 h at 100 °C and 18 h at 40 °C and 70 %RH, but at the end of the cooling period, an additional 20 mL of deionized water was added to a selection of the samples to simulate overhydration. After ten cycles, the samples hydrated by deliquescence alone gave an average total yield of glycine oligomers of 16.0%, while the samples subjected to additional hydration by deionized water gave an average yield of 1.9% (Fig. 3). The addition of extra water better simulates what would be expected of hydration by rainstorms, which unlike rehydration by deliquescence, cannot be counted on to provide a limited amount of water. The extra water reduces yields of glycine oligomerization by increasing the duration of time the oligomers are subjected to hydrolysis and decreasing the duration of time in the cycle where condensation is favorable, after the water has evaporated. While the heat and reduced water activity during the evaporative dry phase support peptide synthesis, heat applied to an overhydrated sample—with higher water activity—can favor hydrolysis. The liquid deliquescent brine already had water activity low enough to support peptide condensation, but the evaporation of samples to dryness was critical to the growth of longer oligomers. Control samples of glycine in liquid deliquescent brines heated in capped vials—and hence, not allowed to evaporate—produced trace yields of Gly 2 and Gly 3 over 10 cycles (1.1% total), but the yields were comparatively lower than the samples allowed to evaporate to dryness (16.0% of Gly ≥2 , with up to Gly 13 observed, see Supplementary Fig. 31).

Fig. 3 The advantage of limited rehydration in wet–dry cycling. Yields of glycine oligomers (excluding DKP) in the presence of (K+, Na+)(Cl−, OH−) after 1, 2, 3, 5, and 10 cycles. Each cycle was 24 h. For the samples marked with triangles, one cycle included 18 h at 40 °C and 70 %RH, followed by 6 h at 100 °C. The samples marked with circles were exposed to the same environmental cycles, but 20 mL of water was added to the mixture before each drying period. This addition simulated heavy rain and overhydration of the sample to verify a shortcoming of the standard model for wet–dry cycling that is obviated by self-regulated, limited rehydration through deliquescence. The error bars represent 95% confidence intervals (n = 3 identical experiments). Source data are provided as a Source Data file Full size image

Deliquescence controlled by alkali salts

Once we established the efficacy of deliquescence to rehydrate the chloride system, we sought to vary the deliquescent salts used in the mixtures to see if the results of atmospheric rehydration in wet–dry cycling are general. Phosphate salts especially piqued our interest for their importance to biology. At 50 %RH, K 2 HPO 4 is deliquescent, while Na 2 HPO 4 is not. While the availability of phosphate salts was likely limited on the Prebiotic Earth41, there is no question that, at some point, phosphate anions became relevant to the development of life42.

In this experiment, rather than vary the relative humidity, we varied the salt. Samples of glycine mixed with K 2 HPO 4 or Na 2 HPO 4 were subjected to ten cycles of 20 h at 40 °C and 50 %RH and 4 h at 120 °C. The results are summarized in Fig. 4. The potassium samples, which were deliquescent and rehydrated completely during the cooling periods, produced total yields of Gly n of 21.2%. The non-deliquescent sodium samples remained solid through all ten cycles and gave a total yield of Gly n of 3.5%. Figure 4b is a histogram tracking the yield of each oligomer measured at the end of cycles 1, 2, 3, 5, and 10 (see Supplementary Figs. 10, 11 and Supplementary Tables 10, 11 for more details). It is apparent that not only do the total yields improve over time for the deliquescent system, but that the distribution of products favors longer oligomers over time. For the prebiotic condensation of amino acids, this feature is presumably advantageous, as longer oligomers are better suited to developing the secondary and tertiary structure observed in modern functional proteins. Even shorter peptides may have served as functional biomolecules on Prebiotic Earth43,44,45.

Fig. 4 Glycine oligomerization in K 2 HPO 4 vs. Na 2 HPO 4 . a The total yields of glycine oligomers (excluding DKP) in the presence of K 2 HPO 4 (red diamonds) or Na 2 HPO 4 (blue circles) after 1, 2, 3, 5, and 10 cycles. Each cycle was 24 total hours: 20 h at 40 °C and 50 %RH followed by 4 h at 120 °C. The error bars represent 95% confidence intervals (n = 3 identical experiments). b Distribution of yields by oligomer length after cycles 1, 2, 3, 5, and 10 in the sample prepared with K 2 HPO 4 . Source data are provided as a Source Data file Full size image