Nucleobases bind to and stabilize aggregates of a prebiotic amphiphile, providing a viable mechanism for the emergence of protocells

Abstract

Primordial cells presumably combined RNAs, which functioned as catalysts and carriers of genetic information, with an encapsulating membrane of aggregated amphiphilic molecules. Major questions regarding this hypothesis include how the four bases and the sugar in RNA were selected from a mixture of prebiotic compounds and colocalized with such membranes, and how the membranes were stabilized against flocculation in salt water. To address these questions, we explored the possibility that aggregates of decanoic acid, a prebiotic amphiphile, interact with the bases and sugar found in RNA. We found that these bases, as well as some but not all related bases, bind to decanoic acid aggregates. Moreover, both the bases and ribose inhibit flocculation of decanoic acid by salt. The extent of inhibition by the bases correlates with the extent of their binding, and ribose inhibits to a greater extent than three similar sugars. Finally, the stabilizing effects of a base and ribose are additive. Thus, aggregates of a prebiotic amphiphile bind certain heterocyclic bases and sugars, including those found in RNA, and this binding stabilizes the aggregates against salt. These mutually reinforcing mechanisms might have driven the emergence of protocells.

Keywords: fatty acids; micelles; nucleosides; origin of life; vesicles.

Fig. 1.

Decanoic acid aggregates selectively bind heterocyclic nitrogenous bases. (A) Structures of purines and pyrimidines tested for interactions with decanoic acid aggregates. Diaminopurine contains an amine at the 2-position in addition to the 6-position as in adenine; 2-aminopurine, also tested in some experiments, has an amine only at the 2-position. Amine substituents are indicated in red. (B) Adenine dialyzes more slowly from a decanoic acid solution than from an acetic acid solution. (Left) Results of a representative experiment in which adenine, at 15 mM, diffused from either 180 mM decanoic acid or from 180 mM acetic acid. Aliquots of dialysis buffer were collected at indicated times and assayed for adenine by measuring absorbance at 260 nm. The rate of release was 24 ± 5% lower from decanoic acid (P < 0.05). (Right) Results of a corresponding representative control experiment with uracil in place of adenine. The rate of release was 8 ± 7% greater, not lower, from decanoic acid (P > 0.05). (C) The presence of 10 mM adenine in a subphase of PBS increases the surface pressure of a Langmuir monolayer of stearic acid. Measurement uncertainty is ±1 mN/m. Stearic acid (18 carbons) was used instead of decanoic acid because the latter does not form a stable Langmuir monolayer. (D) Nucleobases are retained with decanoic acid micelles during ultrafiltration. A solution of 180 mM decanoic acid and each base at 0.03 mM (for purines) or 0.3 mM (for pyrimidines) was partially centrifuged through a 3-kDa–cutoff filter. These concentrations optimize both the percentage of base retained by micelles and the detection of base by absorbance; adenine was evaluated at both 0.3 and 0.03 mM to enable comparison of all the bases. Values are averages, and error bars represent average deviations. (The difference between the means for cytosine and uracil is significant based on Student t test: P = 0.028 by a one-tailed test and 0.056 by a two-tailed test.)

Fig. 2.

Nucleobases and sugars inhibit flocculation of decanoic acid induced by salt. (A) Adenine reduces reflocculation of decanoic acid, and enables vesicle formation, after dissolution of flocs by heat. Test tube solutions of 80 mM decanoic acid/pH 7.65, without and with 30 mM adenine, were treated as indicated. Corresponding samples for microscopy contained 10 μM rhodamine 6G as a dye and were heated and cooled to the indicated temperatures on the microscope stage. Scale bars are 20 μm. (B) Incubation with adenine at 32 °C reduces preexisting flocs. Shown is 80 mM decanoic acid/pH 7.6, with and without 25 mM adenine and 300 mM NaCl as indicated, before and after incubation at 32 °C for 8 h. (The larger volume used for the preincubation set was chosen arbitrarily.) To quantitate the effect, aliquots were incubated in a 96-well plate in parallel, and turbidity was measured after 8 h; the presence of adenine reduced absorbance at 490 nm by 74 ± 1% (average of duplicates). (C) Nucleobases inhibit salt-induced flocculation of decanoic acid. The main panel shows the percent reduction in absorbance of a solution of 80 mM decanoic acid/300 mM NaCl (compared with controls with no base added) vs. the concentration of adenine, in the plate-based assay for flocculation described in the text; results are representative of three experiments. (Inset) Percent reduction in absorbance of a solution of 80 mM decanoic acid/300 mM NaCl containing 7.5 mM adenine, cytosine, or uracil (compared with controls with no base added); error bars represent average deviations of duplicate samples. (D) The inhibition of salt-induced flocculation of decanoic acid by nitrogenous bases correlates with their binding to decanoic acid micelles. The purines (Left) were tested in the plate-based assay for flocculation at 2.5 mM, and the pyrimidines (Right) were tested at 10 mM; samples were run in duplicate, and error bars represent average deviations. Values for percent retained with micelles are from the filtration assay run with bases at 0.3 mM. (E) Sugars inhibit salt-induced flocculation of decanoic acid. The main panel shows the percent reduction in absorbance of a solution of 80 mM decanoic acid/300 mM NaCl (compared with controls with no sugar added) vs. the concentration of sugar, in the plate-based assay for flocculation; the results are representative of three experiments. (Inset) Percent reduction with 90 mM sugar; error bars represent average deviations of duplicate samples.