Small-molecule uptake in membrane-free peptide/nucleotide protocells

The spontaneous phase separation of peptide/nucleotide droplets in water produces membrane-free chemically organized micro-compartments that offer new opportunities for the construction of synthetic cells and development of protocell models of prebiotic organization. Certain small molecules can be sequestered into the droplet interior but the uptake mechanisms are unexplored. Using confocal fluorescence microscopy, P-31 NMR spectroscopy, fluorescence spectroscopy and lateral molecular force microscopy, we probe the molecular interactions associated with sequestration of the water-soluble fluorescent anionic dye 1-anilinonapthalene-8-sulphonic acid (ANS) into positively charged oligolysine/ATP coacervate micro-droplets. Our results indicate that uptake of ANS proceeds initially through electrostatic interactions involving a ternary ANS/oligolysine/ATP complex, followed by a secondary mechanism based on non-polar interactions between ANS and ATP. We demonstrate that at very high levels of ANS the hybrid droplets develop a thin outer shell that is mechanically more compliant than the droplet interior, and acts as a quasi-membrane for restricting the influx of methylene blue. Our results suggest that understanding the mechanisms of molecular uptake into coacervate droplets could provide an important step towards the rational design of molecularly crowded microscale dispersions that display complex fluid behavior, compartment-mediated functionality and primitive aspects of synthetic cellularity.