Construction
of protocells with hierarchical structures and living
functions is still a great challenge. Growing evidence demonstrates
that the membraneless organelles, which facilitate many essential
cellular processes, are formed by RNA, protein, and other biopolymers
via liquid–liquid phase separation (LLPS). The formation of
the protocell in the early days of Earth could follow the same principle.
In this work, we develop a novel coacervate-based protocell containing
membraneless subcompartments via spontaneous liquid–liquid
phase separation by simply mixing single-stranded oligonucleotides
(ss-oligo), quaternized dextran (Q-dextran), and poly(l-lysine)
(PLL) together. The resulting biphasic droplet, with PLL/ss-oligo
phase being the internal subcompartments and Q-dextran/ss-oligo phase
as the surrounding medium, is capable of sequestering and partitioning
biomolecules into distinct regions. When the droplet is exposed to
salt or Dextranase, the surrounding Q-dextran/ss-oligo phase will
be gradually dissociated, resulting in the chaotic movement and fusion
of internal subcompartments. Besides, the external electric field
at a lower strength can drive the biphasic droplet to undergo a deviated
circulation concomitant with the fusion of localized subcompartments,
while a high-strength electric field can polarize the whole droplet,
resulting in the release of daughter droplets in a controllable manner.
Our study highlights that liquid–liquid phase separation of
biopolymers is a powerful strategy to construct hierarchically structured
protocells resembling the morphology and functions of living cells
and provides a step toward a better understanding of the transition
mechanism from nonliving to living matter under prebiotic conditions.
The
membraneless organelles (MLOs) and coacervates of oppositely
charged polyelectrolytes are both formed by liquid–liquid phase
separation. To reveal how the crowded cell interior regulates the
MLOs, we chose the coacervates formed by peptide S5 and single-stranded
oligonucleotide (ss-oligo) at 1:1 charge ratio and investigated the
phase separation processes in polyacrylamide (PAM) and poly(ethylene
oxide) (PEO) media at varying concentrations. Results show that the
droplet formation unit is the neutral primary complex, instead of
individual S5 or ss-oligo. Therefore, the coacervation process can
be described by the classic theory of nucleation and growth. The dynamic
scaling relationships show that S5/ss-oligo coacervation undergoes
in sequence the heterogeneous nucleation, diffusion-limited growth,
and Brownian motion coalescence with time. The inert crowders generate
multiple effects, including accelerating the growth of droplets, weakening
the electrostatic attraction, and slowing down or even trapping the
droplets in the crowder network. The overall effect is that both the
size and size distribution of the droplets decrease with increasing
crowder concentration, and the effect of PEO is stronger than that
of PAM. Our study provides a further step toward a deeper understanding
of the kinetics of MLOs in crowded living cells.
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