Coacervate-pore complexes for selective molecular transport and dynamic reconfiguration

Abstract

Despite surging interests on liquid-state coacervates and condensates, confinement within solid-state pores for selective permeation remains an unexplored area. Drawing inspiration from nuclear pore complexes (NPCs), we design and construct coacervate-pore complexes (CPCs) with regulatable permeability. We demonstrate universal CPC formation across 19 coacervate systems and 5 pore types, where capillarity drives the spontaneous imbibition of coacervate droplets into dispersed or interconnected pores. CPCs regulate through-pore transport by forming a fluidic network that modulates guest molecule permeability based on guest-coacervate affinity, mimicking NPC selectivity. While solid constructs of NPC mimicries are limited by spatial fixation of polymer chains, CPCs of a liquid nature feature dynamic healing and rapid phase transitioning for permeability recovery and regulation, respectively. Looking forward, we expect the current work to establish a basis for developing liquid-based NPC analogs using a large pool of synthetic coacervates and biomolecular condensates.

Fig. 1. Schematic illustrations of nuclear pore complexes (NPCs) and their mimicries of solid or liquid natures.

a The molecular machinery of an NPC featuring a central channel filled with FG-rich proteins to act as a molecular sieve. b Two solid constructs based on surface-grafted brushes and crosslinked polymers, where the spatial fixations are highlighted by purple staples. c A liquid construct based on coacervate-pore complexes (CPCs). d The liquid coacervates are made of small molecules (red and blue), polyelectrolytes (orange and green), or proteins (yellow and purple). e Inhabitation of coacervate droplets into a pore of diameter (Φ) is a spontaneous process as driven by the capillary pressure (P_c), given that the contact angle (θ) is smaller than 90 °. The Laplace pressure (P_L) has a minor effect. f Four features of some CPCs.

Fig. 2. The formation and liquidity of coacervate 1/pore 1 complexes (denoted as COA1@POR1).

a Self-deployment of coacervate droplets into the solid-state pores. b COA1 droplets suspended in the dilute phase as observed in the transmission (left) and fluorescence channel (right). c POR1 on PC membranes as imaged by optical microscopy (left) and SEM (right). d A SEM picture of dried COA1@POR1 membrane, demonstrating the solid remaining in the pores (highlighted by arrows). e Z-stack scan of a COA1@POR1 membrane where the cross sections in the xy, xz, and yz planes are laid out on the left and the 3D reconstruction is presented on the right (box size = 70 × 70 × 7 μm, color bar indicates height in z direction). f The contact angle (θ) of a COA1 droplet on PC surface underneath its dilute phase is 38 °. g The compositions of free COA1 droplets and in-pore COA1 are similar. Data are presented as mean ± SD (n = 3 independent experiments). h The FRAP curves for COA1@POR1, COA10@POR1, and COA17@POR1, and their corresponding free droplets. Sequential snapshots of a FRAP process of COA1@POR1 from −4 s (before bleaching) to 25 s of recovery, where the irradiation area is highlighted by a white circle. The coacervates in (b, e, h) are fluorescently labeled by 5 μM RhB. Scale bars = 20 μm (b) and 10 μm (c–e, h).

Fig. 3. Molecular structures of the coacervate systems and porous membranes.

Molecular structures of the coacervate constituents (a–c) and the membranes (d), where the positive and negative charges are highlighted by blue and red circles, respectively. The structure of FUS-MBP is generated by AlphaFold2, whose prediction on the intrinsically disordered region (IDR) may not be accurate. e SEM pictures of bare pores (POR3 to POR5) and optical images of the pores loaded with COA1 in the transmission and fluorescent channels, where COA1 is mixed with 5 μM RhB. Scale bars = 50 μm.

Fig. 4. Selective permeation of CPCs.

a A microscope-compatible, permeability device with a lower chamber, a CPC membrane, and an upper chamber. b A scheme showing the guest molecules in the feed phase tend to traverse the COA10@POR2 to reach the receiving phase. c False-color, fluorescence images to indicate the disfavored partitioning of PEG-20K (MW ~ 20 K, 0.04 g L^–1), PEG-5K (MW ~ 5 K, 0.04 g L^–1), BSA (MW ~ 65 K, 5 μM), RhB (MW = 479, 5 μM), and ε-PL (MW ~ 5 K, 0.01 g L^–1) into the COA10 droplets (4 g/L, 0.2 M NaCl, pH = 7.2). Two PEGs are tagged by HEMA-RhB, BSA by TRITC, and ε-PL by FITC. d Permeation of BSA across the COA10@POR2 membrane following a concentration gradient from the lower to the upper chambers. e The decay of normalized fluorescence intensity in the feeding phase for five different guests (dots) is fitted by an exponential function (lines) to give the rate constant (R). f Transport rate relative to that across bare pores (R/R₀) is plotted against the K values, where a liner fitting can reasonably match the data (black line). Data are presented as mean ± SD (n = 3 independent experiments). The blue, cyan, green, orange, and red in (e, f) correspond to PEG-20K, PEG-5K, BSA, RhB, and ε-PL as the guest molecules, respectively. Scale bars = 30 μm (c) and 5 μm (d).

Fig. 5. Facilitated transportation of two cargoes by two designer carriers.

a A conjugated carrier, PSBZB (MW ~ 20 K)-complementary DNA (cDNA, MW ~ 6.4 K), binds the cargo, ssDNA (MW ~ 6.4 K), to form a complex. b Fluorescence images showing that the cargo (1 μM ssDNA) is rejected while the carrier and complex are enriched by the COA10 droplets (4 g/L, 0.2 M NaCl, pH = 7.2). FAM indicates the fluorescent tag, carboxyfluorescein. FRET (Förster resonance energy transfer) channel confirms the formation of complex and its enrichment to the droplets. c Decay of normalized fluorescence intensity for the transportation of the cargo across the bare pores (red dots), the cargo across COA10@POR2 (blue dots), and the complex across COA10@POR2 (green dots); exponential fittings are given by lines. Comparisons between the cargo and complex on the transport rate (normalized by the bare pore rate (d) and on the partitioning ratios (e). f A conjugated carrier, PSBZB-G4 (DNA G-quadraplex), binds the cargo, ThT, to form a complex. g Optical images of the transmission and fluorescence channels showing that the cargo (1 μM ThT, MW = 319) is rejected by the COA10 droplets (4 g/L, 0.2 M NaCl, pH = 7.2) and fluorescently dim and that the complex is enriched by the droplets and fluorescently bright. The fluorescence decay (h), normalized transport rates (i) and partitioning ratios (j) similar to panels (c–e), except that the cargo is now ThT. Scale bars = 50 μm (b, g). Data are presented as mean ± SD (n = 3 independent experiments, d, i).

Fig. 6. Dynamic reconfiguration of CPCs.

a Schematic illustration of the phase transitioning of COA17 to a dissolved or a solid state, regulating the CPC permeability. b Optical observation of phase transitions of free COA17 droplets into a dissolved state at pH = 2 and into a solid state at pH = 13. c Correspondingly, the in-pore coacervates reach a dissolved, liquid, and solid states at acidic, neutral, and basic conditions as confirmed by optical images in the transmission and fluorescence channels and by a SEM picture. d Fluorescence decay curves for 5 μM RhB and 0.1 g L^–1 PEG-5K to pass the COA17@POR1 membranes at pH = 13 (blue), 7 (green), and 2 (red), respectively. The lines are exponential fittings. The insets compare the transportation rates normalized by the rate at pH = 7. Data are presented as mean ± SD (n = 3 independent experiments). e Schematic drawings and optical images of the transmission and fluorescence channels for a COA17@POR1 membrane at its pristine, punctuated, and healed states, respectively. Scale bars = 50 μm (b) and 10 μm (c, e). The coacervates in (c, e) are fluorescently labeled by a trace amount of TRITC-BSA.