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.


Materials and instruments
Solvents and reagents were obtained from commercial sources and used without further purification unless otherwise stated. Porphyrin 1, protoporphyrin IX mono t-butyl ester, was synthesized according to the procedure reported in the literature. [1] Each compound, mono-substituted protoporphyrin IX or protoheme IX, 1-3 and Hemeazide, also forms a similar mixture of two regioisomers. All amine-modified and dye-labeled ssDNA strands were obtained HPLC-pure from IDT Integrated DNA Technologies, whereas all unlabeled DNA strands were obtained purified by using a desalting column. Upon arrival, all DNA strands were dissolved in milliQ in DNA Lo binding tubes (Eppendorf) to reach a final concentration of 0.1-1 mM. Myoglobin (M0630) and Horseradish Peroxidase (77332) were purchased from Sigma-Aldrich. ESI-TOF MS analyses were performed with a micrOTOF-II mass spectrometer (Bruker). 1 H NMR spectra were collected on an AVANCE III HD (400 MHz) NMR spectrometer (Bruker). The 1 H NMR chemical shift values are reported in ppm relative to a residual solvent peak.

Expression and purification of p-azido-phenylalanine (Y151pAzF) eYFP using amber stop codon suppression
The gene for enhanced yellow fluorescent protein (eYFP) was available in a pET28a expression vector with a Cterminal hexahistidine tag, and kindly provided by Remco Arts and Maarten Merkx. The native tyrosine at position 151 was mutated to encode for an amber stop codon for incorporation of p-azidophenylalanine, using the QuikChange Lightning Multi Site-Directed Mutagenesis kit (Agilent), according to the manufacturer's instructions (using primer 5'-ACTACAACAGCCACAACGTCTAGATCATGGCCGAC-3') The plasmid encoding for eYFP-Y151pAzF was co-transformed into E. coli BL21(DE3) competent bacteria together with a pEVOL plasmid containing an engineered orthogonal amino acyl tRNAse/tRNA pair from M. janaschii (a kind gift from Peter Schultz, Addgene plasmid #31186). [3], [4] The bacteria were cultured at 37°C in 200 mL LB medium, 25 μg/mL kanamycin and 25 μg/mL chloramphenicol. Protein expression was induced at OD600=0.6 by addition of 1 mM β-D-1thiogalactopyranoside and 0.02% (w/v) arabinose. Simultaneously, p-azido-L -phenylalanine was added directly to the culture medium at a final concentration of 1 mM. Expression was carried out in the dark for ~16 h at 30°C. Cells were harvested by centrifugation at 10,000 g for 10 min at 4°C and lysed by resuspending the pellet in BugBuster (5 mL/g pellet, Merck) supplemented with Benzonase nuclease (25 U per 10 mL buffer) for 45 min on a shaking table at room temperature. The soluble fraction (cleared lysate) containing protein was collected by centrifugation at 40,000 g for 30 min at 4°C. Purification was performed by Ni 2+ -affinity chromatography using the C-terminal hexahistidine tag. This also removed protein fragments truncated at position 151, which is typically seen as a result of unsuccessful amber codon suppression. The cleared lysate was loaded on a Ni-charged column ( After centrifugation (14,000 g for 30 min at 4 °C) the supernatant was removed, and the pellet was dissolved in 100 µL of 1xPBS (pH 7.4). This procedure was repeated once and after centrifugation the pellet was washed with 95% ice-cold ethanol (v/v, in water). The mixture was centrifuged again (14,000 g for 15 min at 4 °C) and the pellet was lyophilized and stored at -30°C.

Preparation of eYFP-ssDNA conjugates
Conjugation reactions were performed on a 100 μL scale using 30 μM protein and 90 µM BCN-ssDNA in 100 mM potassium phosphate buffer, pH 7.2. The reaction mixture was incubated for 4 h at RT in the dark under continuous shaking at 800 rpm. Unreacted ssDNA and unreacted protein were removed by size exclusion column chromatography (Superdex 200 10/300 preparative column (GE Healthcare), eluent: 10 mM potassium phosphate buffer + 100 mM NaCl), and ion-exchange chromatography respectively. After equilibration of the ion-exchange column (0.5 mL strong anion-exchange spin columns, Thermo Scientific) with purification buffer (10 mM KPi, pH 7.0), the protein mixture was directly loaded onto the column in 400 μL fractions, according to the manufacturer's instructions. Elution was performed by a stepwise increase in NaCl concentration in the buffer (100 mM to 600 mM). eYFP-ssDNA conjugates eluted between 400 and 500 mM NaCl and were characterized by SDS-PAGE ( Figure  S1C). Elution fractions containing pure eYFP-ssDNA conjugates were pooled and the concentration was determined by measuring the absorption at 513 nm.

Preparation and characterization of ssDNA introduced-, and dye labeled HRP and Mb
Synthesis of Heme-azide Scheme S1. Synthesis of Heme-azide. Each protoporphyrin IX derivative contains a structural isomer due to the modified position at the 6-or 7-propionate side chain.

Preparation of reconstituted HRP and Mb with Heme-azide
Removal of heme from horse radish peroxidase (HRP) and myoglobin (Mb) was performed using Teale's method as follows. A solution of proteins (50 M as a monomer) in 100 mM of L-histidine solution at 4 ˚C was acidified to pH 2 by addition of 1 M aqueous HCl. Unbound heme was extracted with 2-butanone (5 times) and the colorless aqueous solution was neutralized by dialysis with 100 mM potassium phosphate buffer, pH 7.0, at 4˚C. A DMSO solution of heme-azide (2 mM, 200 L) was added to a solution of obtained apoprotein (30 M, 10 mL) in 100 mM potassium phosphate buffer, pH 7.0. After gently shaking overnight at 4 ˚C, excess heme-azide was removed with a HiTrap desalting column. Concentrations of the obtained Mb and HRP were determined by measuring the absorption at 408 nm and 403 nm assuming an extinction coefficient of 1.8×10 5 M -1 cm -1 and 1.0×10 5 M -1 cm -1 , respectively.

Preparation of ssDNA and Dye introduced HRP and Mb
Conjugation reactions were performed on a 100 μL scale using 30 μM HRP-heme-azide or Mb-heme-azide and 90 µM BCN-ssDNA in 100 mM potassium phosphate buffer, pH 7.2. The reaction mixture was incubated for 4 h at RT, whilst shaking at 800 rpm. Unreacted ssDNA was removed by size exclusion column chromatography (Superdex 200 10/300 preparative column (GE Healthcare), eluent: 100 mM potassium phosphate buffer). Cy3-NHS and Cy5-NHS (1 µL, 10 mM stock solution in dry DMSO) were added to the protein solution (10 µM, 200 µL) and gently shaken overnight at 4 ˚C in the dark. After adding 2 eq of complementary uptake strand and incubating for 30 min at 4˚C, unreacted dye and excess uptake strand were removed by size exclusion column chromatography (Superdex 200 10/300 preparative column (GE Healthcare), eluent: 100 mM potassium phosphate buffer, Fig S2).

Confocal microscopy
Images were acquired using a Leica TCS SP8 confocal laser scanning microscope equipped with a white light laser, using a HCX PL APO CS 63.0x 1.20 NA water immersion objective and HyD detector. The pinhole was set to 1 Airy Unit (111 μm). Single plane images of 1024x1024 pixels were acquired with a scan rate of 600 Hz, and line averaged 4 times. The respective channels were recorded sequentially using the following parameters:

Image analysis
Confocal images were analyzed with FIJI (ImageJ). Fluorescence distribution between artificial cell populations was determined using standard ImageJ functions. Particles were manually selected with the elliptical selection tool, size and mean pixel intensity were determined with the measure tool. For each analysis >10 particles were included. For quantification, mean grey values were normalized to the maximum grey value of the image that was used for comparison (time point 0 for time measurements, or before addition of the releaser strand for release studies). Figure S1. Conjugation of ssDNA to eYFP (pAzF) using strain-promoted alkyne-azide cycloaddition. A) Reaction scheme for the modification of 5'-amine modified C6 ssDNA with bicyclononyne-N-succinimidyl carbonate (BCN-NHS). B) Reaction scheme for the modification of eYFP (pAzF) with BCN modified ssDNA. Reactions were performed for 4h at room temperature (RT) in phosphate-buffered saline (PBS), pH7.4. C) SDS-PAGE analysis of the conjugation reaction of BCN-ssDNA (4.2 kDa) to eYFP (30.5 kDa) and purification by anion exchange chromatography. eYFP modified with ssDNA appeared as a band with a higher mass than eYFP and eluted between 400 and 500 mM NaCl.          First REL B-20 (orange) was added (second column) and 12nt-ssDNA-Mb was released, then REL A-40 (dark blue) was added and 12nt-ssDNA-HRP was released (third column). Finally, REL C-20 (pink) was added and 12nt-ssDNA-eYFP was released (right column). Incubation time after adding each REL strands was 30 minutes. The experiment was performed in PBS containing 5 mM MgCl2, pH 7.4, I = 185 mM. Yellow is eYFP; Pink is Cy3; and Cyan is Cy5. Scale bars represent 20 µm. Figure S12. Schematic and confocal overview images showing retention of 12nt-ssDNA-Mb-Cy3, 12nt-ssDNA-HRP-Cy5 and 12nt-ssDNA-eYFP inside the coacervates after 60 minutes when omitting all corresponding releaser strands. The experiment starts when 12nt-ssDNA-Mb, 12nt-ssDNA-HRP and 12nt-ssDNA-eYFP are cosequestered inside the coacervates following hybridization with a complementary uptake strand (left column). The 12nt-ssDNA handle b conjugated to Mb hybridized with UPT B-20 (light green/dark green), 12nt-ssDNA handle a conjugated to HRP hybridized with UPT A-40 (light purple/red), and 12nt-ssDNA handle c conjugated to eYFP hybridized with UPT C-20 (light blue/purple). After 60 minutes incubation in the absence of releaser strands at room temperature all the proteins remain inside the coacervate core (right). The experiment was performed in PBS containing 5 mM MgCl2, pH 7.4, I = 185 mM. Yellow is eYFP; Pink is Cy3; and Cyan is Cy5. Scale bars represent 20 µm. Figure S13. Schematic and confocal images showing the presence and absence of exchange between two artificial cell populations with or without addition of the releaser strand REL A-20 (blue). The sender population was loaded with a histidine-tagged 12nt-ssDNA-eYFP and uptake strand (UPT A-20), the receiver population was equipped with NTA-amylose, Ni 2+ and Cy5 labeled succinylated BSA (Cy5-BSA). The experiment starts at 0 min when 12nt-ssDNA-eYFP is uptaken inside the coacervate core following hybridization with UPT A-20 (middle column). Addition of the releaser strand (REL A-20) triggers the release of 12nt-ssDNA-eYFP from the sender population. Subsequentially, 12nt-ssDNA-eYFP is sequestered by the receiver population via complex formation between the Histidine tag and Ni 2+ /NTA-amylose (left column). After 60 minutes incubation at room temperature in the absence of releaser stand, no exchange of 12nt-ssDNA-eYFP from the sender to the receiver population has occurred (right column). The experiment was performed in PBS containing 5 mM MgCl2 and 3.75 µM NiCl2, pH 7.4, I = 185 mM. Yellow is eYFP; Red is succinylated Cy5-BSA. Scale bars represent 20 µm.