Phase separation in crowded micro-spheroids: DNA–PEG system

Abstract

Living cells are characterized as micro-vesicles encapsulating highly crowded macromolecular media, including DNA. To probe the intrinsic property of such crowded systems in a micro-closed environment, we observed a mixture of DNA and hydrophilic polymer, PEG within an aqueous micro-spheroid. It was found that the mixture causes phase-segregation by depositing DNA near the spheroid surface, whereas corresponding bulk solution sustains isotropic phase. After the phase segregation, DNA molecules in the condensed phase exhibit time evolution to an ordered state. This surface-mediated segregation is discussed in terms of the depletion effect under micro-confinement.

Introduction

Living cells maintain their lives under highly concentrated conditions of macromolecules such as proteins and nucleic acids along with other small molecules [1]. As for an example, in Escherichia coli, typical total mass density of protein and nucleic acid within the cytoplasm is ca. 220 mg/ml, indicating highly crowded condition [2]. In contrast, most of the biochemical and biophysical studies on the reaction mechanisms, kinetics and thermodynamics of proteins and nucleic acids have been carried out in vitro using dilute aqueous solutions of purified biopolymer constituents [3]. Thus, it is of considerable importance to examine how DNA molecules behave in a crowded solution of a confined cell-sized space ranging ∼1–100 μm. As a typical manifestation of crowding effect with macromolecules, we may consider the general tendency of de-mixing, or phase-segregation, due to an entropic interaction, i.e. depletion effect [4], [5].

It is well known that DNA condenses in the presence of hydrophilic polymer and salt such as NaCl and KCl [6] which is called polymer salt induced (psi) condensation [7], [8]. The hydrophilic solvable polymer, polyethylene–glycol (PEG) has been frequently adapted for the study of psi condensation [9], [10]. It has been revealed that single long DNA molecules undergo discrete transition between elongated coil and compact globule state in the presence of PEG and salt [11]. Whereas in concentrated DNA solutions with PEG, DNA chains tend to take elongated conformation by aligning parallel to each other, being similar to liquid crystal structures [12].

In the present Letter, we focus our attention on the change of the state of DNA molecules under crowded condition under confinement to micrometer length scales. We adapted the experimental system of a cell-sized droplet entrapping DNA and PEG. In order to make the experimental conditions to be simpler, we studied relatively short DNA molecules (300–700 bps) which behave as elastic rods. We show that phase-segregation is generated in a confined environment for the macromolecular solution, whereas segregation is not caused in the bulk solution, under the same composition of the solution. This initial study is a first step towards understanding of phase segregation of macromolecular solutions under confinement.

In relation to the specific effect of confinement on the segregation of DNA in a crowded micro-environment, it may of interest to note that condensed part of genomic DNA, i.e., heterochromatin, is often localized underneath the nuclear membrane, suggesting the similar effect as was observed in the present Letter [13], [14].