Fig 1.
Experimental systems to study protein-DNA co-condensation.
A Schematic of experimental setup wherein the ends of a single DNA molecule (blue) are tethered to the glass slide using biotin-streptavidin interactions. Proteins (green) phase-separate with DNA to form protein-DNA co-condensates. B Schematic of the optical tweezers assay: a DNA molecule (blue) is held between two optically trapped beads (black) via biotin–streptavidin interactions.
Table 1.
Model parameters.
Fig 2.
DNA sequence dictates the position of the condensates.
A Schematic represents a homogeneous DNA model; each monomer (blue) binds to proteins (green) with an identical binding affinity (2 kBT). B Schematic represents heterogeneous DNA I model; monomers (red) at the center bind proteins with an affinity of 2.25 kBT, whereas monomers (yellow) at the periphery have an interaction strength of 1.75 kBT. Proteins bind to each other with an interaction strength of 2 kBT. C, D Snapshots of DNA configurations for homogeneous and heterogeneous DNA I, in the absence of proteins, are shown. E, F Snapshots of protein-DNA co-condensates in equilibrium for the two models are shown. To generate the plots, we fixed the normalized end-to-end distance () and varied
. G, H Probability distribution of DNA and proteins along the longitudinal axis at
= 0.6, for different bulk protein concentrations (
), shown for homogeneous and heterogeneous DNA I, respectively. For each
value, data from five independent replicates are included in the plots. I, J Representative kymographs for position of condensates along the contour as a function of time for homogeneous DNA (
=0.6,
= 109.9
) and heterogeneous DNA I (
=0.6,
= 93.01
) are shown.
Fig 3.
Average interfacial DNA affinity dictates capillary forces.
A and B Capillary force is plotted as a function of at
=84.50
and as a function of
at
=0.6 for homogeneous (salmon) and heterogeneous DNA I (purple), respectively. Each point in the plots represents the average of five independent replicates, with error bars denoting the standard deviation. The snapshots show condensates for heterogeneous DNA I where red represents high-affinity monomers while yellow represents low-affinity monomers. Proteins are shown in green. Snapshots (left to right) show condensates for the following parameter sets: (i)
= 0.2 and
= 84.50
, (ii)
= 0.8 and
= 84.50
, (iii)
= 0.6 and
= 42.25
and (iv)
= 0.6 and
= 126.75
.
Fig 4.
DNA sequence heterogeneity leads to the co-existence of multiple protein-DNA condensates at equilibrium.
A Schematic represents heterogeneous DNA II model: two blocks of high-affinity monomers (2.25 kBT, 100 monomers in red) are separated by a block of low-affinity monomers (0.1 kBT, grey) of identical length. Proteins (green) interact with each other with an interaction strength of 2 kBT. B Snapshots shown for cases with two condensates co-existing at equilibrium for all five independent realizations at and
=84.50
. High-affinity monomers, low-affinity monomers, and proteins are shown in red, grey, and green respectively. C Scaled radius of the larger droplet (black) and smaller droplet (red) is shown for all the five trajectories quantifying the arrested coarsening. D Representative kymograph showing visualization of coarsening kinetics for one initial condition.
Fig 5.
Sequence heterogeneity in partial -DNA engenders multiple condensates as metastable states.
A Schematic represents the Partial -DNA: Eleven kinds of monomers (yellow to maroon) are introduced, based on AT content of 10 base pairs, modeled as one monomer. The monomer-protein binding affinities vary in the range of 0.1 to 4 kBT depending on the AT content of the corresponding 10 bp motif that represents the monomer (see the model section). Proteins (green) bind to each other with an affinity of 2 kBT. B Snapshots shown for five independent realizations at
and
=84.50
(top to down). C, D Representative kymograph showing visualization of coarsening kinetics for replicate 2 and 5. E Scaled radius of the larger condensate (black) and smaller condensate (red) is shown for replicate 2 which leads to a single condensate at equilibrium. F Scaled radius of the larger condensate (black) and smaller condensate (red) is shown for replicate 5 which shows coexistence of two condensates as a kinetically trapped state. G Bar plots show the average interfacial affinity (maroon) and volume (orange) for two condensates observed in the case of replicates 1, 4, and 5.
Fig 6.
Local interfacial DNA affinity dictates global capillary forces.
A Capillary force as a function of at fixed
=0.6. B Capillary force as a function of
at fixed
. C (top panel) Snapshots of condensates at concentrations marked as (i), (ii), and (iii) in A. Monomers are shown in yellow to red in ascending order protein binding affinity and the proteins are shown in green. C(bottom panel) Bar plots represent monomer-protein binding affinities. The grey-shaded region shows the interface. The red curve shows the probability of the monomer being inside the condensate. D capillary force correlates with average interfacial affinities. Grey points show the capillary forces and interfacial affinities for 30 individual replicates at the six
values in A. The purple points show the mean and standard deviation at each
. The red dashed line is a fit to the mean data.
Fig 7.
A comparison between simulation and experimental results.
Scatter plot shows the number of condensates as a function of end-to-end distance for Dps (taken from Figure 4 of [63]), Sox2 (taken from Figure 1b and 3i of [10]), and HP1 (taken from Figure 4B of [26]). The dashed line, depicting the condensate number of unity, reflects sequence-independent co-condensation, whereas the region shaded in pink shows sequence-dependent co-condensation of protein and DNA.
Fig 8.
Key insights from simulations of DNA sequence-dependent protein–DNA Co-condensation.
First column Cartoon representations of the four DNA models considered in our study. Second column Probability of each monomer being inside the condensate is plotted as a function of monomer position along the contour for a shorter homogeneous DNA (Nm = 50, = 0.8, and
= 84.5
), heterogeneous DNA I (
= 0.6 and
= 84.5
), heterogeneous DNA II (
= 0.6 and
= 84.5
), and partial
-DNA (
= 0.2 and
= 84.5
). Third column Representative kymographs are shown for all the models for the same conditions as in the second column. Fourth column Coarsening kinetics of a single final condensate is shown for five independent realizations of homogeneous DNA (Nm = 500,
= 0.6, and
= 126.75
) and heterogeneous DNA I (
= 0.6 and
= 126.75
). Coarsening kinetics of two droplets (larger condensate radius in grey and smaller condensate radius in red) are shown for heterogeneous DNA II (
= 0.6 and
= 84.5
), and partial
-DNA (
= 0.6 and
= 126.75
). Panels are arranged as homogeneous DNA, heterogeneous DNA I, heterogeneous DNA II, and Partial
-DNA from top to bottom.