Enzymatic degradation of liquid droplets of DNA is modulated near the phase boundary

Abstract

Biomolecules can undergo liquid–liquid phase separation (LLPS), forming dense droplets that are increasingly understood to be important for cellular function. Analogous systems are studied as early-life compartmentalization mechanisms, for applications as protocells, or as drug-delivery vehicles. In many of these situations, interactions between the droplet and enzymatic solutes are important to achieve certain functions. To explore this, we carried out experiments in which a model LLPS system, formed from DNA “nanostar” particles, interacted with a DNA-cleaving restriction enzyme, SmaI, whose activity degraded the droplets, causing them to shrink with time. By controlling adhesion of the DNA droplet to a glass surface, we were able to carry out time-resolved imaging of this “active dissolution” process. We found that the scaling properties of droplet shrinking were sensitive to the proximity to the dissolution (“boiling”) temperature of the dense liquid: For systems far from the boiling point, enzymes acted only on the droplet surface, while systems poised near the boiling point permitted enzyme penetration. This was corroborated by the observation of enzyme-induced vacuole-formation (“bubbling”) events, which can only occur through enzyme internalization, and which occurred only in systems poised near the boiling point. Overall, our results demonstrate a mechanism through which the phase stability of a liquid affects its enzymatic degradation through modulation of enzyme transport properties.

Targeted modulation of protein liquid–liquid phase separation by evolution of amino-acid sequence

“…Biomolecular condensates, or membraneless organelles, such as stress granules [3], P-granules [4,5], the nephrin-NCK-WASP system [6] and the nucleoli [7], are formed by LLPS and have diverse biological functions. LLPS inside cells plays a very diverse range of roles beyond membraneless compartmentalisation, such as in gene silencing via heterochromatin formation [8][9][10], in gene activation by facilitating the formation of super-enhancers [11], in buffering cellular noise [12], in modulating enzymatic reactions [13] and in sensing pH changes in the skin [14]. However, some biomolecular condensates emerge spontaneously inside cells without as-yet clearly identified functions; it has been hypothesised that some of these might be implicated in the emergence of phase-separation-related pathologies [15].…”

CRISPR-based DNA and RNA detection with liquid-liquid phase separation

“…Furthermore, we recently showed that the elongation of short RNA oligomers with a template-independent RNA polymerase (polynucleotide phosphorylase) causes the onset of LLPS, and that this can be reversed by enzymatic RNA degradation (27). Finally, Saleh et al have shown that phase-separated droplets consisting of DNA nanostars can be degraded using a DNA restriction enzyme (36). However, a direct experimental test of length-dependent LLPS in mixtures of nucleic acids and polycations is lacking.…”

The role of liquid–liquid phase separation in regulating enzyme activity

“…The mechanism behind such proximity-based activity increase is likely also tied to intrinsic biophysical properties of the condensate. Saleh et al investigated circumstances where the phase-separated component consists of the substrate (DNA) whereas the surrounding dilute phase contains the enzyme (a restriction enzyme -SmaI) free in solution [78]. Enzymatic activity, and thus degradation of the droplet, was heightened by those enzymes which penetrated within the droplet.…”