Uncharged Components of Single-Stranded DNA Modulate Liquid–Liquid Phase Separation With Cationic Linker Histone H1

Abstract

Liquid–liquid phase separation (LLPS) of proteins and DNAs has been recognized as a fundamental mechanism for the formation of intracellular biomolecular condensates. Here, we show the role of the constituent DNA components, i.e., the phosphate groups, deoxyribose sugars, and nucleobases, in LLPS with a polycationic peptide, linker histone H1, a known key regulator of chromatin condensation. A comparison of the phase behavior of mixtures of H1 and single-stranded DNA-based oligomers in which one or more of the constituent moieties of DNA were removed demonstrated that not only the electrostatic interactions between the anionic phosphate groups of the oligomers and the cationic residues of H1, but also the interactions involving nucleobases and deoxyriboses (i) promoted the generation of spherical liquid droplets via LLPS as well as (ii) increased the density of DNA and decreased its fluidity within the droplets under low-salt conditions. Furthermore, we found the formation of non-spherical assemblies with both mobile and immobile fractions at relatively higher concentrations of H1 for all the oligomers. The roles of the DNA components that promote phase separation and modulate droplet characteristics revealed in this study will facilitate our understanding of the formation processes of the various biomolecular condensates containing nucleic acids, such as chromatin organization.

Phase-separation propensity of non-ionic amino acids in peptide-based complex coacervation systems

“…S2a for representative peptides: K7Y3 with aromatic side chains, K7S3 with hydrophilic side chains, and K7L3 with aliphatic side chains). Considering previous reports, 6,21,22,30,31 this behaviour confirmed that the observed condensates were not solid aggregates but liquid droplets with highly fluidic properties. To further investigate the liquid properties of the droplets, we carried out fluorescence recovery after photobleaching (FRAP) experiments, a method commonly used to measure the mobility of molecules inside droplets.…”

“…π-π Interactions between the side chain of aromatic amino acids and nucleobases contribute to the formation of droplets between proteins and nucleic acids. 21,22,40 The nucleobases are embedded in the dsDNA of dR15 and its complementary strand (dsDNA15) due to base pairing, thus preventing access of the peptides to the nucleobases. Therefore, we assumed that dsDNA15 could be used to examine the effect of the nucleobases.…”

“…This is one of the mechanisms thought to underlie various membrane-less organelles and biomolecular condensates in eukaryotic cells [17][18][19] and is often the model of choice for protocell studies. 3,20 We have also recently used this LLPS mechanism to understand the role of the structural features of DNAs in LLPS, 21,22 to elucidate the compartmentalization of enzymes into droplets and its effect on sequential enzyme reactions, 23 and to facilitate the preparation of high-concentration protein solutions for use in protein formulations. 24,25 Here, as scaffolds for complex coacervation, we selected cationic peptides modified with various non-ionic amino acids and DNA of different sequences.…”

A sePARate phase? Poly(ADP-ribose) versus RNA in the organization of biomolecular condensates

“…Phase behavior also differs between single-stranded and double-stranded sequences. High LLPS potential of unfolded single-stranded nucleic acids with exposed nucleobases is increased by contributing π-electron–related interactions ( 54 ). These include π–π and cation–π interactions of the nucleobase components ( 54 ) and sugar–π contacts enabled by deoxyribose moieties ( 55 ).…”

“…High LLPS potential of unfolded single-stranded nucleic acids with exposed nucleobases is increased by contributing π-electron–related interactions ( 54 ). These include π–π and cation–π interactions of the nucleobase components ( 54 ) and sugar–π contacts enabled by deoxyribose moieties ( 55 ). In contrast, double-stranded DNA has higher charge density than single-stranded DNA, and binds more strongly to polycations, thereby forming solid aggregates (at low ionic strength) or a liquid–crystalline phase (at high ionic strength), while remaining base-paired ( 53 ).…”