Enzymatic Reaction Network-Driven Polymerization-Induced Transient Coacervation

Abstract

A living cell has a highly complex microenvironment whereas numerous enzyme-driven processes are active at once. These procedures are incredibly accurate and efficient, although comparable control has not yet been established in vitro. Here, we design an enzymatic reaction network (ERN) that combines antagonistic and orthogonal enzymatic networks to produce adjustable dynamics of ATP-fueled transient coacervation. Using horseradish peroxidase (HRP)-mediated Biocatalytic Atom Transfer Radical Polymerization (BioATRP), we synthesized poly(dimethylaminoethyl methacrylate), which subsequently formed coacervates with ATP. We rationally explored enzymatic control over coacervation and dissolution, using orthogonal and antagonistic enzyme pairs viz., alkaline phosphatase, Creatine phosphokinase, hexokinase, esterase, and urease. ATP-fuelled coacervates also demonstrate the enzymatic catalysis to prove its potential to be exploited as a cellular microreactor. Additionally, we developed ERN-polymerization-induced transient coacervation (ERN-PIC), with complete control over the system, polymerization, coacervation, and dissolution. Notably, the coacervation process itself determines functional properties, as seen in selective cargo uptake. The strategy offers cutting-edge biomimetic applications, and insights into cellular compartmentalization by bridging the gap between synthetic and biological systems. The development of temporally programmed coacervation is promising for the spatial arrangement of multienzyme cascades, and offers novel ideas on the architecture of artificial cells.

Keywords: ATP; BioATRP; Coacervates; Enzymatic Reaction Network; Liquid-liquid phase separation.

Scheme 1

Schematic illustration of enzymatic reaction networks (ERNs) driving in situ polymer synthesis using aqueous BioATRP, time‐controlled complex coacervation with ATP, and dissolution. The coacervates also depict microreactor behavior and selective cargo encapsulation.

Figure 1

(a) Scheme for PDMAEMA synthesis through BioATRP. (b) Molar mass distribution of polymers P1 and P2 synthesized in two different batches. (c) Turbidity measurements for titration of PDMAEMA with ATP and ADP. (d) CLSM images of complex coacervates of PDMAEMA with ATP, [Resorufin]=0.5 mol %, scale bar=2 μm. (e) FRAP kinetics. (f) Corresponding CLSM images obtained during FRAP of PDMAEMA with 5 mM ATP coacervate, [Nile red]=0.5 mol %, scale bar=5 μm. [PDMAEMA]=0.5 mM, 50 mM HEPES, pH 7.3

Figure 2

(a) Enzymatically mediated stimuli responsive coacervation. Kinetics of the pH‐modifying enzymes. Arrows indicate the addition of substrates. [PDMAEMA]=1.25 mM, [ATP]=8 mM, Esterase=200 U/mL, Urease=285 U/mL, [ethyl acetate]=240 mM, [urea]=24 mM, and 4.5 pH, 10 mM acetate buffer. (b) ATP‐driven ALP‐mediated transient coacervation of PDMAEMA. Time dependent % turbidity changes with varied units of ALP. [PDMAEMA]=0.5 mM, [ATP]=5 mM, 50 mM HEPES, pH 7.3. (c) ERN‐mediated ATP‐driven transient coacervation of PDMAEMA (HK, and CPK). Time‐dependent % Turbidity changes. [PDMAEMA]=0.5 mM, [ATP]=[ADP]=5 mM and 4 mM, CPK=20 U/mL, HK=10 U/mL, [PCr]=7.5×10⁻³ M, [Glucose]=0.1 M, 50 mM HEPES and pH 7.3. N=3,±standard deviation.

Figure 3

CLSM images of coacervates with segregated protein/enzymes. (a) coacervates with CPK‐488, BSA‐TAMRA, HK‐647, and overlay. (b) coacervates with urease‐488, BSA‐TAMRA, GOX‐647, and overlay. Scalebars=20 μm. (c) Scheme of bioPIC process. (d) % Turbidity of the monomer, and polymer after reaction (along with unreacted monomer), filtered polymer to remove unreacted monomer on coacervation (n=3,±standard deviation). (e) The molecular weight distribution of PDMAEMA produced via bioPIC with a photograph of the turbid suspension after polymerization. (f) Scheme of the ERN‐PIC, (g) Time dependent changes in % Turbidity of in the presence, and absence of 0.4 U/mL ALP. (f) CLSM images of coacervates with segregated protein/enzymes, coacervates as black regions around urease‐488, BSA‐TAMRA, GOX‐647, and overlay. Insets are zoomed‐in images. [DMAEMA]=1.5 M, [ATP]=44 mM (bioPIC), 22 mM (ERN‐PIC). Scalebars=20 μm.