Membraneless compartments, such as complex coacervates, have been hypothesized as plausible prebiotic micro-compartments due to their ability to sequester RNA; however, their compatibility with essential RNA World chemistries is unclear. We show that such compartments can enhance key prebiotically-relevant RNA chemistries. We demonstrate that template-directed RNA polymerization is sensitive to polycation identity, with polydiallyldimethylammonium chloride (PDAC) outperforming poly(allylamine), poly(lysine), and poly(arginine) in polycation/RNA coacervates. Differences in RNA diffusion rates between PDAC/RNA and oligoarginine/RNA coacervates imply distinct biophysical environments. Template-directed RNA polymerization is relatively insensitive to Mg2+ concentration when performed in PDAC/RNA coacervates as compared to buffer, even enabling partial rescue of the reaction in the absence of magnesium. Finally, we show enhanced activities of multiple nucleic acid enzymes including two ribozymes and a deoxyribozyme, underscoring the generality of this approach, in which functional nucleic acids like aptamers and ribozymes, and in some cases key cosolutes localize within the coacervate microenvironments.

Phase separation of aqueous solutions containing polyelectrolytes can lead to formation of dense, solute-rich liquid droplets referred to as coacervates, surrounded by a dilute continuous phase of much larger volume. This type of liquid–liquid phase separation is thought to help explain the appearance of polyelectrolyte-rich intracellular droplets in the cytoplasm and nucleoplasm of extant biological cells and may be relevant to protocellular compartmentalization of nucleic acids on the early Earth. Here we describe complex coacervates formed upon mixing the polycation poly­(allylamine) (PAH, 15 kDa) with the anionic nucleotides adenosine 5′-mono-, di-, and triphosphate (AMP, ADP, and ATP). Droplet formation was observed over a wide range of pH and MgCl2 concentrations. The nucleotides themselves as well as Mg2+ and RNA oligonucleotides were all extremely concentrated within the coacervates. Nucleotides present at just 2.5 mM in bulk solution had concentrations greater than 1 M inside the coacervate droplets. A solution with a total Mg2+ concentration of 10 mM had 1–5 M Mg2+ in the coacervates, and RNA random sequence (N54) partitioned ∼10 000-fold into the coacervates. Coacervate droplets are thus rich in nucleotides, Mg2+, and RNA, providing a medium favorable for generating functional RNAs. Compartmentalization of nucleotides at high concentrations could have facilitated their polymerization to form oligonucleotides, which preferentially accumulate in the droplets. Locally high Mg2+ concentrations could have aided folding and catalysis in an RNA world, making coacervate droplets an appealing platform for exploring protocellular environments.

Most RNA folding studies have been performed under non-physiological conditions of high concentrations (≥10 mM) of Mg2+free, while actual cellular concentrations of Mg2+free are only ~1 mM in a background of greater than 50 mM Mg2+total. To uncover cellular behavior of RNA, we devised cytoplasm mimic systems that include biological concentrations of amino acids, which weakly chelate Mg2+. Amino acid-chelated Mg2+ (aaCM) of ~15 mM dramatically increases RNA folding and prevents RNA degradation. Furthermore, aaCM enhance self-cleavage of several different ribozymes, up to 100,000-fold at Mg2+free of just 0.5 mM, indirectly through RNA compaction. Other metabolites that weakly chelate magnesium offer similar beneficial effects, which implies chelated magnesium may enhance RNA function in the cell in the same way. Overall, these results indicate that the states of Mg2+ should not be limited to free and bound only, as weakly bound Mg2+ strongly promotes RNA function under cellular conditions.