Polyamine/Nucleotide Coacervates Provide Strong Compartmentalization of Mg²⁺, Nucleotides, and RNA

Abstract

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.

Directed Growth of Biomimetic Microcompartments

“…[40,[48][49][50] Moreover, enzymes, which partition into the microdroplets, maintain activity within the highly charged and crowded interior. [40,[48][49][50] Moreover, enzymes, which partition into the microdroplets, maintain activity within the highly charged and crowded interior.…”

Bridging the gap betweenin vitroandin vivoRNA folding

“…These droplet phases are another indicator that early life could have arisen in non-membranous compartments. More recently we made coacervates from nucleotides and poly(allylamine) that contain molar concentrations of Mg 2+ and nucleotides, which could facilitate RNA catalysis in an early life scenario (Frankel et al, 2016). …”

Dynamic Synthetic Cells Based on Liquid–Liquid Phase Separation

“…In PEG/dextranA TPSs, single-stranded RNA [57] and double-stranded (ds) DNA [58] preferentially accumulate in the dextran phase, with increased partition coefficients reported for longerp olynucleotides. [56] Whereas dissociation of nucleic acidd uplexesw as reported in protein-based droplets, [63] the co-sequestration of complementary oligonucleotides in surfactant-based protocells was found to promote base pairing. Rapid exchange of RNA oligomers, for instance, was reported in aP EG/dextranA TPS and in polylysine/ATP coacervate droplets, [59] yet very little exchange of single-stranded (ss) DNA oligomers was observed for up to 48 hi nabinary poly(diallyldimethylammonium bromide) (PDDA)/ATP coacervate droplet population produced by microfluidics.…”

“…These favourable properties are exploited by living cells to achiever apid regulationo fb iochemical reactions, and might have potentiali mplications in the emergence of functional protocells capable of selectiveb inding, chemical reactivity (e.g.,c atalysis, protometabolism) and genetice volution (e.g.,s ynthesiso fo ligonucleotides from mononucleotides). [7] In contrast, complexc oacervate droplets have been found to sequester small solutes,i ncluding ions, [56] through complementary electrostatic interactions, together with hydrophobic effects associated with the lower dielectric constant of the droplets' interiors relative to the dilute phase. This differential solute sequestration is quantified experimentally by measuring an equilibrium partitionc o-efficient, usually defined as the solute concentrationw ithin the droplets divided by the solute concentration in the continuous phase.…”

“…This differential solute sequestration is quantified experimentally by measuring an equilibrium partitionc o-efficient, usually defined as the solute concentrationw ithin the droplets divided by the solute concentration in the continuous phase. [21,52,56] In one notable example, Koga et al demonstrated the formation of coacervate microdroplets from nucleoside triphosphates (ATP), diphosphates (ADP,F AD, NAD) or monophosphates (AMP) mixed with oppositely charged oligolysine chains,and reported a20-fold increase in ATPc oncentration relative to the dilute continuous phase. [55] Low-molecular-weight solutes do not usually exhibit high levels of partitioning in neutral polymer ATPSs.…”