Designing DNA-based cytoskeletons for synthetic cells

Discover how Zhan and colleagues used microfluidics and DNA nanotechnology to build a functional cytoskeleton for synthetic cells.

This research summary describes how Pengfei Zhan, Kevin Jahnke, Na Liu, and Kerstin Göpfrich built a synthetic functional cytoskeleton using DNA nanotechnology in a cell-sized microfluidic system.

Their work was recently featured in Nature Chemistry and shows promising results towards building synthetic cells from bottom-up.

Abstract

The cytoskeleton controls shape, internal organization, cargo transport, and other essential cell functions. Mimicking such features of natural cytoskeletons is key to designing functional synthetic cells. Zhan and colleagues used DNA nanotechnology and microfluidics to build a synthetic cell-sized system of a functional DNA-based cytoskeleton. The system consists of DNA tiles self-assembled into filament networks operating in a microfluidic compartment. Their artificial cytoskeleton showed features of their natural counterparts, such as ATP-triggered polymerisation and reversible assembly. Additionally, their results indicated autonomous cargo transport along DNA filaments. Overall, this work highlights the great potential of DNA nanotechnology and microfluidics for building synthetic cells from the bottom up.

Introduction | Cytoskeleton architecture for synthetic cells

The cytoskeleton consists of a dynamic cytoplasmic network of interconnected protein filaments that extends from the cell nucleus to the cell membrane. It provides mechanical support, controls cell shape, and is involved in signal transduction and intracellular transport of molecules.

Designing cytoskeletons to perform these diverse and vital functions is essential to creating cell-free synthetic cells from the bottom up. Yet, despite rapid advances in DNA nanotechnology, constructing multi-functional cytoskeletons remains challenging.

In this paper, Zhan and colleagues describe how they designed a DNA-based cytoskeleton operating in cell-sized microfluidic compartments. Their synthetic cytoskeleton mimics their natural counterparts in essential functions such as compartmentalisation, ATP-triggered polymerisation, and reversible assembly. Their data also suggests intracellular cargo transport, a key process of cell physiology.

Aims

• To build a functional DNA-based cytoskeleton in a microfluidic compartment using DNA nanotechnology.
• To master the required complexity of cytoskeleton function, which is a crucial step towards cell-free synthetic cellular architecture.

Experiment setup | DNA filament design and encapsulation

DNA-based filaments were synthesised from pre-determined sequences (supplementary Table 1), mixed in a Tris-EDTA-based buffer, and annealed in a thermocycler. The DNA filaments were visualised via transmission electron microscopy (TEM) and atomic force microscopy (AFM). 

The DNA tiles were self-assembled into filament networks and encapsulated in surfactant-stabilized water-in-oil droplets produced by an OB1 flow controller and a PDMS chip. The oil phase (1.4vol%) consisted in dissolving a PEG-based fluorosurfactant in HFE 7500 oil. Because the OB1 flow controller delivers nearly any flow and pressure profile to a system, it is ideal for droplet formation and particle encapsulation.

The content to be encapsulated was suspended in the water phase at varying conditions:

• For polymerisation of DNA tiles functionalized with ATP: ATP-sensitive tiles (in a Mg2+ containing buffer) were mixed with 10mM ATP and encapsulated into droplets using microfluidics. 

• For assembly and disassembly of the DNA tiles: for disassembly, DNA filaments and ATP were mixed, and 10µM invader strands for (de-)polymerisation were added to the solution, followed by immediate microfluidic droplet encapsulation. Adding 37.5µM anti-invader strands right before microfluidic encapsulation, caused the DNA filaments to assemble back together. 

• For actin encapsulation: actin monomers were labelled with 20µL AB DTT DD (double density) buffer and 3.3µL of rhodamine-phalloidin (13U), followed by immediate droplet encapsulation using microfluidics and imaged during polymerisation.

Both oil and water phases were injected into the PDMS chip channels via a 0.4×0.9mm polytetrafluoroethylene tubing. An inverted microscope connected to the microfluidic setup was used to observe the droplet formation and the encapsulation process (Figure 1).

To mimic active vesicle transport along microtubules by cytoskeleton motor proteins, RNA overhangs were added to the DNA filaments to serve as transport tracks. Gold nanoparticles were used as inorganic cargo, small unilamellar vesicles (SUV) served as organic, and both types of particles were attached to a complementary DNA sequence matching the RNA overhangs (for more details, refer to Methods).

Materials

• OB1 Pressure controller
• PDMS – chip (Sylgard 184, Dow Corning)
• PEG-base fluorosurfactant (Ran Biotechnologies)
• HFE-7500 oil (DuPont)
• Polytetrafluoroethylene tubing (Bola)
• Axio Vert.A1 (Carl Zeiss)
• DNA strands (Sigma-Aldrich) (the DNA sequences are available in the supplementary data of the paper)
• RNA-DNA conjugate strands (Integrated DNA Technologies)
• Tris-EDTA buffer (see paper)
• Confocal fluorescence microscope LSM 880 or LSM 900 (Carl Zeiss)
• AFM (Molecular Imaging, Bruker Technologies)
• Please refer to the paper for further details on equipment, calculations, solutions, and buffers.

Key findings | DNA-based cytoskeleton structure and function

Zhan and colleagues successfully designed a cell-sized microfluidic system consisting of multi-functional DNA tiles that undergo dynamic assembly and disassembly inside water-in-oil droplets.

Synthetic cytoskeleton design

Each DNA tile contains five DNA strands forming a micrometre-long hollow DNA tube with four sticky ends that serve as binding domains and form tubular DNA filaments (Figure 2).

Their observations also indicate the autonomous transport of lipid-based vesicles or gold nanoparticles along the filaments of the synthetic cytoskeleton. Ribonuclease H (RNase H)-mediated hydrolysis powers up the supposedly guided transport of lipid membrane vesicles or gold nanoparticles along the filaments (Figure 2).

Confocal microscopy shows the monodisperse droplets containing the assembled DNA filaments. Figure 3 and Video 1 confirm the dynamism and constant rearrangement of the DNA filaments inside water-in-oil droplets.

Video 1: DNA filaments confined inside water-in-oil microfluidics droplets showing dynamic remodelling and rearrangement. The droplet size can be engineered to fit different experimental conditions and requirements. (Video taken from Zhan, P., Jahnke, K., Liu, N. et al. Nat. Chem. 2022).

DNA filament assembly and disassembly

The DNA tiles are reversibly disassembled by a dual-responsive DNA filament that contains an NCL-specific aptamer triggering the assembly and an ATP-specific aptamer triggering disassembly. This dynamic polymerisation process inside the droplet was quantified by confocal microscopy (Figure 4 and Video 2).

Video 2: Polymerization of ATP-aptamer-modified DNA filaments inside microfluidics water-in-oil droplets. (Video taken from Zhan, P., Jahnke, K., Liu, N. et al. Nat. Chem. 2022).

The polymerisation of the DNA-based filaments inside the droplets (Video 2) increases until 40 min when it reaches a dynamic state. Similarly, the actin filaments (Video 3) polymerise at a comparable rate reaching a similar polymerisation degree (Figure 4b).

Video 3: Polymerization of actin filaments inside microfluidics water-in-oil droplets. (Video taken from Zhan, P., Jahnke, K., Liu, N. et al. Nat. Chem. 2022).

Cargo transport along DNA filaments

Cytoskeleton motor proteins in the microtubules of cells are responsible for active vesicle transport, a vital role in cell physiology. To mimic this feature, Zhan and coworkers designed DNA filaments with RNA overhangs to transport cargo along the DNA strands. Small unilamellar vesicles (SUV) acted as organic cargo, while gold nanoparticles provided insight into inorganic particle transport.

RNase H powered the rolling of the SUV along the DNA filaments via the hybridisation of RNA and DNA molecules (Figures 5 and 6). Transmission electron microscopy confirmed the binding and transport of the SUV along the DNA filament (Video 4).

Video 4: SUV transport along DNA filaments powered by RNase H inside microfluidics water-in-oil droplets. (Image modified from Zhan, P., Jahnke, K., Liu, N. et al. Nat. Chem. 2022).

Gold nanoparticles provided insight into transport mechanisms beyond biological cargo. The results suggest that these particles also move along the DNA filaments powered by RNase H inside the droplets (Figure 7).

Conclusion | Synthetic cells at the interface between technology and biology

This study highlights DNA nanotechnology’s and microfluidics’ great potential to replicate living systems and build synthetic cells from the bottom up. However, the main challenge is still replicating some of the multi-functional features of living cells – such as intracellular transport, communication, and organisation – in synthetic replicas.

The polymerisation of the DNA tiles containing split ATP aptamers upon adding ATP provides a higher biological significance to the synthetic model described in this paper. Also, although much slower than in living cells, the suggested inorganic gold nanoparticles and lipid vesicles transport along the DNA filaments inspire future investigation.

“A challenging but insightful experiment would be monitoring the cargo transport along the DNA-based filaments on the single cargo level within the compartment”, suggest the authors. “The transport rate of vesicles on microtubules in living cells is still much faster”, they highlight. 

Microfluidics encapsulation processes have paved the way for mimicking living cells. In this study, the sequential addition of molecules after encapsulation of DNA filaments into water-in-oil droplets was successfully achieved by picoinjection, fusion, or light-triggered release of caged compounds. 

“Along the route, we may engineer synthetic cells at the interface between technology and biology for applications in biomedicine, robotic drug delivery, nanomachinery, artificial cellular signalling and communications, and beyond ” conclude the authors. 

This article was written by Thais Langer.