Temperature-Responsive Peptide–Nucleotide Coacervates

[Image: see text] Coacervates are a type of liquid–liquid phase separated (LLPS) droplets that can serve as models of membraneless organelles (MLOs) in living cells. Peptide–nucleotide coacervates have been widely used to mimic properties of ribonucleoprotein (RNP) granules, but the thermal stability and the role of base stacking is still poorly understood. Here, we report a systematic investigation of coacervates formed by five different nucleoside triphosphates (NTPs) with poly-l-lysine and poly-l-arginine as a function of temperature. All studied combinations exhibit an upper critical solution temperature (UCST), and a temperature-dependent critical salt concentration, originating from a significant nonelectrostatic contribution to the mixing free energy. Both the enthalpic and entropic parts of this nonelectrostatic interaction decrease in the order G/A/U/C/T, in accordance with nucleobase stacking free energies. Partitioning of two dyes proves that the local hydrophobicity inside the peptide–nucleotide coacervates is different for every nucleoside triphosphate. We derive a simple relation between the temperature and salt concentration at the critical point based on a mean-field model of phase separation. Finally, when different NTPs are mixed with one common oppositely charged peptide, hybrid coacervates were formed, characterized by a single intermediate UCST and critical salt concentration. NTPs with lower critical salt concentrations can remain condensed in mixed coacervates far beyond their original critical salt concentration. Our results show that NTP-based coacervates have a strong temperature sensitivity due to base stacking interactions and that mixing NTPs can significantly influence the stability of condensates and, by extension, their bioavailability.