Atomistic insights into the reentrant phase-transitions in polyuracil and polylysine mixtures

Abstract

The phase separation of protein and RNA mixtures underpins the assembly and regulation of numerous membraneless organelles in cells. The ubiquity of protein-RNA condensates in cellular regulatory processes is in part due to their sensitivity to RNA concentration, which affects their physical properties and stability. Recent experiments with poly-cationic peptide-RNA mixtures have revealed closed-loop phase diagrams featuring lower and upper critical solution temperatures. These diagrams indicate reentrant phase transitions shaped by biomolecular interactions and entropic forces such as solvent and ion reorganization. We employed atomistic simulations to study mixtures with various RNA-polylysine stoichiometries and temperatures to elucidate the microscopic driving forces behind reentrant phase transitions in protein-RNA mixtures. Our findings reveal an intricate interplay between hydration, ion condensation, and specific RNA-polylysine hydrogen bonding, resulting in distinct stoichiometry-dependent phase equilibria governing stabilities and structures of the condensate phase. Our simulations show that reentrant transitions are accompanied by desolvation around the phosphate groups of RNA, with increased contacts between phosphate and lysine side chains. In RNA-rich systems at lower temperatures, RNA molecules can form an extensive pi-stacking and hydrogen bond network, leading to percolation. In protein-rich systems, no such percolation-induced transitions are observed. Furthermore, we assessed the performance of three prominent water force fields-Optimal Point Charge (OPC), TIP4P-2005, and TIP4P-D-in capturing reentrant phase transitions. OPC provided a superior balance of interactions, enabling effective capture of reentrant transitions and accurate characterization of changes in solvent reorganization. This study offers atomistic insights into the nature of reentrant phase transitions using simple model peptide and nucleotide mixtures. We believe that our results are broadly applicable to larger classes of peptide-RNA mixtures exhibiting reentrant phase transitions.

FIG. 1.

Representative simulation snapshots of clusters formed in U₄ and K₄ mixtures as a function of stoichiometry and temperature. Silver represents U₄, and green represents K₄. The RNA–peptide ratios in each column are 3:1, 1:1, and 1:3, with rows corresponding to (a) 270 K, (b) 330 K, and (c) 390 K.

FIG. 2.

Reentrant transitions as a function of U₄:K₄ stoichiometry (3:1, 1:1, and 1:3) and temperature (270, 330, and 390 K). (a) Cluster fraction is quantified as the number of biomolecules in the cluster for the U₄:K₄ ratio at three different temperatures. (b) SASA per residue in clusters. (c) Radial distribution functions defined over atoms of U₄ and K₄. (d) Number of water molecules surrounding the U₄ molecule at 270 K. (f) Number of water molecules surrounding the K₄ molecule at 270 K. (g) Cluster shapes for a 3:1 ratio, (h) for a 1:1 ratio, and (i) for a 1:3 ratio.

FIG. 3.

Mean number of hydrogen bonds for three U₄–K₄ ratios: 3:1, 1:1, and 1:3 at different temperatures: 270, 330, and 390 K. Hydrogen bonds are defined for the following pairs: (a) U₄–U₄, (b) U₄–K₄, and (c) K₄–K₄. (d) Snapshot showing the interaction of K₄ side chain with the phosphate of U₄. (e) Radial distribution function of alpha-carbon of K₄ around the phosphate of RNA for the 1:1 U₄–K₄ ratio at three different temperatures. (f) Snapshot showing the interaction of the base of RNA with K₄. (g) Radial distribution function of alpha-carbon of K₄ with respect to the base of RNA for 1:1 U₄–K₄ ratio at three different temperatures.

FIG. 4.

Dehydration of backbone and bases of RNA. (a) Schematic representation of the U₄–K₄ interaction as a function of increasing temperature. Number of water per (b) phosphate and (c) base of 1:1 U₄–K₄ ratio at three different temperatures.

FIG. 5.

Effect of water model on temperature-dependent cluster transformation. (a) Comparison of Lennard-Jones interaction of TIP4P-2005, TIP4P-D, and OPC water models. Number of water around RNA at different temperatures from (b) TIP4P-D, (c) TIP4P-2005, and (d) OPC. Comparison of cluster shape and size at high temperatures: (e) TIP4P-2005 at 510 K, (f) TIP4P-D at 390 K, and (g) OPC at 510 K.