DNA Local-Flexibility-Dependent Assembly of Phase-Separated Liquid Droplets

Abstract

Phase separation of intracellular components has been recently realized as a mechanism by which cells achieve membraneless organization. Here, we study the associative liquid-liquid phase separation (LLPS) of DNA upon complexation with cationic polypeptides. Comparing the phase behavior of different single-stranded DNA as well as double-stranded DNA (dsDNA) sequences that differ in persistence lengths, we find that DNA local flexibility, not simply charge density, determines the LLPS. Furthermore, in a nucleotide- and DNA-dependent manner, free nucleotide triphosphates promote LLPS of polypeptide-dsDNA complexes that are otherwise prone to precipitation. Under these conditions, dsDNA undergoes a secondary phase separation forming liquid-crystalline subcompartments inside the droplets. These results point toward a role of local DNA flexibility, encoded in the sequence, in the regulation and selectivity of multicomponent LLPS in membraneless intracellular organization.

Figure 1

DNA rigidity due to basepairing is unfavorable for LLPS. (a) The chemical structure of PLL. (b) Phase-separated droplets of PLL and a random sequence 21-nt ssDNA; [NaCl] = 150 mM, N/P ∼1, room temperature. Confocal fluorescence images show homogenous partitioning of both PLL and ssDNA. (c) Coarsened and hollow structures resulting after the addition of a 1:1 equivalent of complementary ssDNA (ssDNA_comp) to preformed PLL-ssDNA droplets shown in (b); resulting N/P ∼0.4. The distribution of PLL and DNA is heterogeneous, with both polymers preferentially partitioned close to the droplet/solution interface. (d) FCS autocorrelation curves are measured for labeled PLL before (τ_transit = 28 ms) and after (τ_transit = 120 ms at the edges and τ_transit = 0.029 ms in the interior of the hollow structures) the addition of ssDNA_comp. The dynamics are significantly reduced at the edges, whereas the interior is near water like. (e) A bright-field image of precipitates of PLL-dsDNA complexes under the same conditions of salt, temperature, and N/P as in (b).

Figure 2

Local flexibility dependence of ssDNA LLPS in the presence of PLL. Bright-field microscopy images of (a) PLL-poly(T) and (b) PLL-poly(A) phases at different NaCl concentrations are shown; N/P ∼1, room temperature. At 500 mM NaCl, poly(A) shows very little phase separation compared to poly(T), attributable to differences in intrinsic persistence lengths.

Figure 3

Local flexibility dependence of dsDNA LLPS in the presence of PLL. Bright-field microscopy images of (a) PLL-poly(GC) and (b) PLL-poly(AT) phases at different NaCl concentrations are shown; N/P ∼1, room temperature. Poly(GC) forms droplets at much lower salt concentrations compared to poly(AT). At higher salt concentrations (830 mM for poly(GC) and 990 mM for poly(AT)), the LLPS gives rise to liquid-crystalline droplets. The striations observed in the droplets are characteristic of cholesteric phases. Inset scale bars, 5 μM.

Figure 4

Role of ATP in LLPS of dsDNA. Bright-field and confocal microscopy images of (a) PLL-poly(GC) and (b) PLL-poly(AT) phases with and without ATP are shown. In the presence of ATP, liquid-like phases are formed; [NaCl] = 150 mM, N/P ∼1 (ATP charge = 1.4 mM). The distribution of ATP inside the liquid-like phases is heterogeneous, giving rise to subcompartmented appearance. These subcompartments are dsDNA-rich liquid-crystalline phases, with the birefringent domains visible under cross-polarizers. Higher concentrations of ATP are required for poly(AT) compared to poly(GC) to form similar droplets (Fig. S10).

Figure 5

Role of UTP in LLPS of dsDNA. Bright-field and confocal microscopy images of (a) PLL-poly(GC) and (b) PLL-poly(AT) phases in the presence of UTP are shown; [NaCl] = 150 mM, N/P ∼1 (UTP charge = 1.4 mM). The transition to liquid-like phases requires higher UTP concentration compared to ATP. At N/P ∼0.2 (UTP charge = 7 mM), dsDNA-rich liquid-crystalline subcompartments are observed, with the birefringent domains visible under cross-polarizers.