The Growth Rate of DNA Condensate Droplets Increases with the Size of Participating Subunits

Abstract

Liquid–liquid phase separation (LLPS) is a common phenomenon underlying the formation of dynamic membraneless organelles in biological cells, which are emerging as major players in controlling cellular functions and health. The bottom-up synthesis of biomolecular liquid systems with simple constituents, like nucleic acids and peptides, is useful to understand LLPS in nature as well as to develop programmable means to build new amorphous materials with properties matching or surpassing those observed in natural condensates. In particular, understanding which parameters determine condensate growth kinetics is essential for the synthesis of condensates with the capacity for active, dynamic behaviors. Here we use DNA nanotechnology to study artificial liquid condensates through programmable star-shaped subunits, focusing on the effects of changing subunit size. First, we show that LLPS is achieved in a 6-fold range of subunit size. Second, we demonstrate that the rate of growth of condensate droplets scales with subunit size. Our investigation is supported by a general model that describes how coarsening and coalescence are expected to scale with subunit size under ideal assumptions. Beyond suggesting a route toward achieving control of LLPS kinetics via design of subunit size in synthetic liquids, our work suggests that particle size may be a key parameter in biological condensation processes.

Light-controlled growth of DNA organelles in synthetic cells

“…Once irradiated with UV light (320 nm) for 30 s, visible condensates form within minutes, as shown in figure 2 c . The average condensate area measured over time (image processing methods are described in the electronic supplementary material, §2) shows that condensates grow by ripening and fusion at a rate comparable to that observed in previous experiments by us and others [9,13]; however, at 30 s irradiation condensates grow at less than half the speed of the control (no hairpin). These experiments validate our hypothesis that condensation of a three-arm DNA nanostar can be controlled by modulating the conformation of one of the arms through a UV-responsive hairpin.…”

“…The condensate growth rate should follow a power law with respect to time, as we showed in previous work [9] (electronic supplementary material, §3.1). This model considers particles that are large with respect to the solvent and assumes that condensate growth is mainly driven by fusion.…”

“…A functional nanostar has three or more arms each including single-stranded palindromic domains at the tip of the arms (sticky ends) that enable the formation of bonds among nanostars and their condensation [5]. The phase transitions of DNA nanostars depend on typical factors like nanostar concentration, temperature and ionic strength of the solvent [6–9]. Their phase diagram can be shaped by the nanostar design parameters, including sticky end length, sequence and number of arms [10,11].…”

“…For DNA condensates to operate as mimics of dynamic cellular organelles, it is necessary to understand and harness the kinetics of their formation and dissolution. The ‘constitutive’ growth rate of DNA condensates is determined by certain structural parameters of the nanostars: for example, longer arms speed up both coarsening and fusion of condensates [9], while longer sticky ends reduce the speed of fusion, as the bond mobility is reduced [11]. To modify condensate formation/dissolution rates over time (without altering the nanostar structural parameters or the environment), a simple approach is to change the amount of particles available for phase separation: this can be done via biochemical reactions that convert nanostars between an active and an inactive form.…”

DNA Droplets: Intelligent, Dynamic Fluid

“…[9,10] In the context of artificial cell studies, DNA droplets have emerged as a promising biomaterial for the construction of intelligent artificial cells (Figure 1A). [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] DNA droplets are micrometer-scale liquid-like condensate composed purely, or at least mostly, of DNA, a biomolecule in which genetic information is encoded. Since the first report published in 2018, [14] several groups have explored DNA droplets to demonstrate their fundamental functions, properties, and advantages, which are not parallel to other biomolecules.…”