DNA-Mediated Protein Shuttling between Coacervate-Based Artificial Cells

Abstract

The regulation of protein uptake and secretion is crucial for (inter)cellular signaling. Mimicking these molecular events is essential when engineering synthetic cellular systems. A first step towards achieving this goal is obtaining control over the uptake and release of proteins from synthetic cells in response to an external trigger. Herein, we have developed an artificial cell that sequesters and releases proteinaceous cargo upon addition of a coded chemical signal: single-stranded DNA oligos (ssDNA) were employed to independently control the localization of a set of three different ssDNA-modified proteins. The molecular coded signal allows for multiple iterations of triggered uptake and release, regulation of the amount and rate of protein release and the sequential release of the three different proteins. This signaling concept was furthermore used to directionally transfer a protein between two artificial cell populations, providing novel directions for engineering lifelike communication pathways inside higher order (proto)cellular structures.

Keywords: Coacervates; DNA; Proteins; Supramolecular Signalling; Synthetic Cells.

Figure 1

Schematic illustrating protein recruitment and release from coacervate‐based artificial cells using DNA strand displacement. A) Liquid–liquid phase separation of oppositely charged cationic quaternized amylose (Q‐am) and anionic carboxymethyl‐amylose (CM‐am) into microdroplets. B) Hierarchical self‐assembly of the terpolymer (PEG‐PCLgTMC‐PGlu) on the droplet surface. C) Preparation of protein–DNA conjugates via BCN–azide click chemistry. The UPT strand was attached to connected‐DNA on proteins. Cy3 and Cy5 dyes were covalently introduced to Mb and HRP, respectively, before introduction of UPT strand. D) Exemplary sequestration of eYFP in the coacervate core following hybridization to a complementary DNA uptake (UPT) strand. E) Release of eYFP from the coacervate matrix triggered by the addition of a displacing releaser (REL) DNA strand.

Figure 2

Dynamic uptake and release of protein inside coacervate artificial cells using DNA strand displacement. A) Schematic and confocal images of the partitioning of 12nt‐ssDNA‐eYFP, 12nt‐ssDNA‐HRP and 12nt‐ssDNA‐Mb (100 nM each) inside the coacervate core following DNA hybridization to a complementary UPT strand. Omitting UPT prevents uptake. Incubation time was 30 minutes at 4 °C. B) Schematic and confocal images of 12nt‐ssDNA‐eYFP (100 nM) release from coacervates triggered by the addition of a Cy3‐labeled release strand (REL, 120 nM). Different time points show different artificial cells. After adding the REL strand, the solution was incubated for 60 minutes at room temperature. C) Box‐plots displaying the quantified fluorescence intensity of eYFP inside the coacervate core. D) Representative confocal images of the cycling of eYFP uptake and release from coacervates following the consecutive addition of UPT and REL strands. Concentrations of 12nt‐ssDNA‐eYFP and UPT strand were 100 nM at UPT1 step. 120 nM REL strand was added at REL1 step, and then 500 nM UPT and 600 nM REL strands were added at each uptaking and releasing steps, respectively. The incubation time at each step was 15 minutes. Different time points show different artificial cells. E) Box‐plots displaying the quantified fluorescence intensity of eYFP inside coacervates during the cycle. All experiments were performed in PBS containing 5 mM MgCl₂, pH 7.4, ionic strength (I)=185 mM. Fluorescence intensity was determined inside the core of≥15 coacervates. ▪ represents the mean, ♦ represents outliers. Scale bars represent 20 μm.

Figure 3

Multiplex control over protein release from coacervate‐based artificial cells. A) Schematic and quantification of the release of 1 equivalent eYFP (100 nM) from coacervate‐based artificial cells following two times addition of 0.5 equivalents REL strand (50 nM) at 20 and 70 minutes (black arrowheads). Graph depicts the decrease in eYFP fluorescence intensity inside coacervates over time. B) Dependence of REL strand complementarity on releasing speed of eYFP. Graph depicts the decrease in fluorescence intensity inside coacervates over time, following the addition of REL strands with complementary bases varying between 12+0 nt (0) and 12+20 nt (20) at room temperature. Fluorescence intensity was determined inside the core of ≥5 coacervates. Error bars represent standard deviation. For uptaking eYFP with each UPT strands, all samples were incubated over 30 minutes before adding each REL strands at 4 °C. C) Schematic and confocal images showing the selective step‐wise release of Mb, HRP and eYFP following the consecutive addition of corresponding REL strands. After adding each REL strand, the solutions were incubated for 30 minutes at room temperature. All experiments were performed in PBS containing 5 mM MgCl₂, pH 7.4, ionic strength (I)=185 mM. Different time points represent different artificial cells. Scale bars represent 20 μm.

Figure 4

Protein shuttling between two different coacervate‐based artificial cell populations. A) Schematic showing exchange between two artificial cell populations. The sender population was loaded with eYFP and UPT strand, the receiver population was equipped with NTA‐amylose and Ni²⁺. Upon addition of REL strands, His‐tagged eYFP was released from the sender population and incorporated by the receiver population. B) Structure of NTA‐amylose. C) Confocal images showing the release and subsequent uptake of eYFP (100 nM) from the sender population (blue) to the receiver population (red). After adding the REL strand, the solution was incubated for 60 minutes at room temperature. The experiment was performed in PBS containing 5 mM MgCl₂ and 3.75 μM NiCl₂, pH 7.4, I=185 mM. D) Box plots displaying the quantified fluorescence intensity of eYFP inside the sender and receiver artificial cell population before and after addition of the REL strand. Fluorescence intensity was determined inside the core of ≥15 coacervates. ▪ represents the mean, ♦ represents outliers. Scale bars represent 20 μm.