NS–PLL Coacervation Dynamics

• NS–PLL Coacervation is the phase separation of negatively charged DNA nanostars and cationic poly-L-lysine driven by cooperative electrostatic and base-pairing interactions.
• Its phase behavior exhibits tunable two-phase and three-phase coexistence modulated by salt concentration, temperature, and macromolecular ratios.
• The system enables programmable compartmentalization and targeted delivery, offering practical applications in synthetic biology and materials science.

NS–PLL Coacervation is the phase separation phenomenon arising in mixtures of negatively charged nanostructures (such as DNA nanostars, NS) and poly-L-lysine (PLL), a cationic polypeptide, in aqueous solvent. The process is governed by the interplay of electrostatic attractions, base-pairing interactions (when NSs exhibit sticky ends), and solution conditions such as temperature and ionic strength. Recent research elucidates a rich landscape of equilibrium and nonequilibrium states, multi-phase coexistence, and tunable structural properties, contributing to both biological understanding and materials science.

1. Molecular Mechanisms: Electrostatic and Associative Interactions

Coacervation in the NS–PLL system is cooperatively driven by two distinct classes of molecular interactions. The first is heterotypic electrostatic attraction between the negatively charged backbone of DNA nanostars and the positively charged ε-amino groups of PLL. Each DNA nanostar can carry substantial negative charge (e.g., –192e), ensuring strong affinity for PLL whenever salt screening is not overwhelming. The second is homotypic base-pairing between palindromic sticky ends of nanostars (for pNS constructs), which can promote self-association and formation of NS-rich droplets even in the absence of PLL.

The interplay of these interactions is highly nontrivial. Elevated salt concentrations reduce the driving force for electrostatic (NS–PLL) coacervation by screening the Coulomb interaction, but simultaneously enhance base pairing by attenuating repulsive interactions between like-charged DNA arms, thus promoting sticky-end hybridization. As a result, NS–PLL coacervation can be stabilized at high salt and high temperature specifically when both interactions cooperate, whereas individually they would not suffice for condensation (Abraham et al., 22 Jul 2025).

2. Phase Behavior: Two-Phase and Three-Phase Coexistence

Systematic variation of salt and temperature reveals a complex phase diagram. In npNS+PLL mixtures (non-palindromic NS without base pairing), the phase behavior is dominated by the electrostatic attraction: there is an upper critical salt concentration beyond which the solution is homogeneous, an intermediate range where coacervate droplets exist, and a low-salt regime where gel-like, arrested states form due to overly strong electrostatics.

In pNS+PLL mixtures (palindromic NS with sticky ends), homotypic base pairing introduces a lower critical threshold for salt. Combining this with the upper threshold of electrostatics results in a broad window for liquid–liquid phase separation and, under certain charge stoichiometries and salt concentrations, regions of three-phase coexistence: pNS-rich droplets (base-pair stabilized), PLL-rich coacervates (electrostatically stabilized), and the surrounding dilute phase. The partitioning of different NS species between these condensed phases enables programmable compartmentalization (Abraham et al., 22 Jul 2025).

3. Kinetics and Nonequilibrium Structures

The kinetic pathways to phase separation depend strongly on the salt concentration and macromolecular composition. At high salt and low free-energy barriers, spinodal decomposition leads to rapid nucleation and growth of liquid droplets, followed by slow coarsening. At lower salt or for certain charge ratios, nucleation barriers induce long-lived nonequilibrium aggregates or gels. Direct observation (fluorescence microscopy) shows that PLL can infiltrate initially NS-rich droplets slowly, with dramatic kinetic differences for short versus long PLL chains. In specific regimes, a phenomenon akin to complete wetting is observed, where a gel-like coacervate layer encapsulates pNS-rich droplets and exchanges mass on timescales of many hours or days (Abraham et al., 22 Jul 2025).

4. Experimental and Theoretical Framework

The NS–PLL coacervation system is characterized experimentally using dual-color fluorescent microscopy with specifically labeled NS and PLL, alongside systematic changes in NaCl concentration (0.1 M to 2.5 M) and annealing protocols for NS. Quantitative metrics are extracted for droplet/dense phase composition and coexistence.

Theoretical modeling employs a mean-field free-energy density of the form

$$f = f_{\rm poly} + f_{\rm micro} + f_{\rm self} + f_{\rm assoc},$$

where $f_{\rm poly}$ includes ideal entropy and excluded volume of macromolecules, $f_{\rm micro}$ accounts for microion entropy and excluded volume, $f_{\rm self}$ models the self-energy of polyions in their electrical double layers (with explicit Debye length $\kappa$ and charge size parameters), and $f_{\rm assoc}$ encodes the base-pair binding free energy via Wertheim’s theory. All terms are parameterized directly from molecular characteristics or established thermodynamic databases. This theoretical approach successfully reproduces qualitative shifts in phase boundaries and equilibria, including the expansion of the coacervation window through the cooperativity of electrostatic and base-pairing interactions (Abraham et al., 22 Jul 2025).

5. Dependence on Solution Conditions

The rich behavior of NS–PLL coacervation is highly sensitive to environmental parameters:

• Salt concentration: Increasing [NaCl] screens electrostatics (destabilizing npNS+PLL droplets) but facilitates base pairing (stabilizing pNS-only droplets). In the dual-interaction system, this leads to coacervate stability at intermediate and even high salt.
• Temperature: Higher temperatures destabilize sticky-end duplexes, leading to an upper critical solution temperature (UCST) for pNS droplets. However, in pNS+PLL mixtures at intermediate salt, droplets can persist across a wide temperature range, only dissolving at very high salt or temperatures exceeding duplex melting.
• Macromolecular stoichiometry: Non-stoichiometric ratios can produce conditions for three-phase coexistence and selective partitioning of NS species in multiphase droplets.
• Kinetic pathway: Sample preparation, annealing protocols, and rate of parameter changes can yield kinetically trapped gels or enable approach to true equilibrium.

6. Functional and Technological Implications

The tunable phase behavior and cooperative interaction mechanisms in NS–PLL coacervation have direct implications for synthetic biology and materials engineering. In particular:

• Compartmentalization: The formation of multiphase droplets with distinct chemical compositions allows programmable spatial separation of biomolecules—reminiscent of natural membraneless organelles.
• Targeted encapsulation and release: The partitioning of NS species between coexisting phases (regulated by salt and temperature) offers routes for sequence-specific or stimulus-responsive cargo delivery.
• Hierarchical material assembly: Layered condensates or core–shell structures can be constructed by mixing NS species with orthogonal sticky ends, giving rise to biomimetic compartmentalization within soft materials.

A plausible implication is that, by systematically varying NS design (e.g., sticky-end sequence, arm length), PLL molecular weight, and solution conditions, one could engineer programmable phase diagrams for bespoke material functions, including reconfigurable microreactors or scaffolds for cell-free biochemical reactions.

7. Theoretical and Practical Advances

The elucidation of NS–PLL coacervation mechanisms underscores several conceptual advances:

• The cooperative/antagonistic interplay between heterotypic electrostatics and homotypic base pairing permits stabilization of condensed phases under conditions where neither alone is sufficient (Abraham et al., 22 Jul 2025).
• Theoretical frameworks combining DLVO-like screening with associating fluid theory parameterized by molecular detail provide quantitative predictions for multiphase coexistence and kinetic pathways.
• The observation of three-phase coexistence and partitioning phenomena reveals a higher level of organizational complexity than standard two-component coacervates.

These findings highlight the broader principle that the integration of orthogonal interaction modes, when combined in single systems, can yield emergent phase behaviors and functionalities not accessible via single interaction types alone. This paradigm is anticipated to be relevant for both the design of bio-inspired materials and the understanding of the compositional complexity of biological condensates.