Short PolyA RNA Homopolymers Undergo Mg2+-Mediated Kinetically Arrested Condensation

Abstract

RNA-RNA interactions have increasingly been recognized for their potential to shape the mesoscale properties of biomolecular condensates, influencing morphology, organization, and material state through networking interactions. While most studies have focused on networking via Watson-Crick base pairing interactions, previous work has suggested a potential for noncanonical RNA-RNA interactions to also give rise to condensation and alter overall material state. Here, we test the phase separation of short polyA RNA (polyrA) homopolymers. We discover and characterize the potential for short polyrA sequences to form RNA condensates at lower Mg²⁺ concentrations than previously observed, which appear as internally arrested droplets with slow polyrA diffusion despite continued fusion. Our work also reveals a negative cooperativity effect between the effects of Mg²⁺ and Na⁺ on polyrA condensation. Finally, we observe that polyrA sequences can act as promoters of phase separation in mixed sequences. These results demonstrate the potential for noncanonical interactions to act as networking stickers, leading to specific condensation properties inherent to polyrA composition and structure, with implications for the fundamental physical chemistry of the system and function of polyA RNA in biology.

Figure 1

PolyrA₂₀ undergoes Mg²⁺-mediated phase separation. (A) PolyrA₂₀ (77 μM, 50 mM Tris, pH 7.5) condenses into spherical droplets in the presence of Mg²⁺, while polyrU₂₀ and polyrC₂₀ do not (scale bars = 5 μm, [Mg²⁺] = 200 mM). (B) Comparatively, no DNA oligomers form spherical condensates under the same conditions. Threshold Mg²⁺ needed to undergo phase separation (C) increases with rA₂₀ concentration and (D) decreases with rA length for a constant amount of polyrA_n (0.5 mg/mL).

Figure 2

PolyrA₂₀ shows slow internal dynamics and mixing (0.5 mg/mL polyrA₂₀, 80 mM Mg²⁺, 50 mM Tris-HCl, pH 7.5). Droplet formation was induced with Mg²⁺ addition separately such that droplets initially were labeled with only one dye labeled species (FAM-rA₂₀ or Cy3-rA₂₀). FAM-labeled droplets were added first to the cover glass. Cy3-labeled droplets were added 15 min after droplet formation, . (A) Fusion of two rA₂₀ droplets from the differentially labeled FAM-rA₂₀ (green) or Cy3-A₂₀ (magenta) populations over the course of 20 min (scale bar = 1.5 μm). The time t = 0 specifies the time just before this fusion event starts. This correlates with a time approximately 7 min after addition of Cy3-A₂₀ labeled droplets and 22 min after droplet formation with Mg²⁺ addition. (B) After 70 min post addition of Cy3-A₂₀ droplets, residual fusion events can be observed by the incomplete mixing of fused droplets. The image is taken at approximately 3 μm above the coverslip from a z-stacked image (Figure S4). (C) Average recovery of normalized FAM-rA₂₀ intensity; the bleached ROI in a FAM-rA₂₀ labeled condensate is shown. The inset depicts a representative droplet at three different time points (prebleached, immediately after bleaching, and at 30 min after bleaching (scale bar = 2 μm)). A line scan through the center of the droplet is shown to the right highlighting the bleach profile.

Figure 3

Cooperative effects of Na⁺ and Mg²⁺ for (A) long polyrU and (B) polyrA₂₀ condensation (50 mM Tris-HCl, pH 7.5; [RNA] = 0.5 mg/mL). The phase boundary shifts toward higher Mg²⁺ with increasing NaCl for polyrA and to lower Mg²⁺ for polyrU.

Figure 4

rN₂₀ + rA₂₀ constructs phase separate and form slowly recovering condensates. (A) Absorption at 350 nm begins to increase around 60 mM MgCl₂ for rA₂₀-tailed constructs ([rN₂₀ + rA₂₀] = 38.6 μM, 50 mM Tris-HCl, pH 7.5), while similar phase separation is not observed in rN₂₀ + rN₂₀ or rN₂₀ + rU₂₀ constructs. (B) Averaged FRAP recovery of partially bleached rN₂₀ + rA₂₀ droplets shows slow and incomplete recovery at 30 min for a small, circular, bleached region of interest. See Figure S6 for individual traces and fits. Images and FRAP represent observations of condensates formed with 200 mM MgCl₂.