Carving the Backbone of the Cell
The Development of a DNA-based Cytoskeleton for Synthetic Cells
Imagine your life without movement. Not just external physical movement, but also movement within the body. Imagine your life without any transportation between cells. Nerve cells not transporting signals to the brain would be just one of many phenomena. Imagine your life without a fixed structure, where every organ, muscle and tissue resulted in a bunch of fluidic mass jiggling around. That is a description of each of your cells without a cytoskeleton.
The National Human Genome Research Institute describes synthetic biology as a field involving “redesigning organisms for useful purposes”. The goal is to change the ‘wiring’ of these organisms so that they develop new abilities that can have applications in a plethora of industries.
In 2010, the JCVI (J. Craig Venter Institute) developed the first cell with a synthetic genome, calling it JCVI-syn1.0. While they did still use the base organelles in the cell of a bacteria, simply replacing the natural DNA with their synthetic version, this marked a key step forward in the field of synthetic biology.
Since then, much has progressed, yet the goal of creating a complete cell from scratch remains unachieved. A key goal for many SynBio institutes has been to replicate a natural cytoskeleton, taking the first step in having the base structure of a synthetic cell. Similar to the role of the skeletal system in humans, the cytoskeleton provides structural support for the cell, along with assisting in motility and transport of different items.
Cytoskeleton
The cytoskeleton is made of three key components : microtubules, intermediate filaments and microfilaments.
Microtubules
Formation of Microtubules — dimers (the union of alpha and beta tubulin) form long chains of polymer sheets, which are rolled up to form a tube.
In a microtubule : one end is anchored in the Microtubule Organizing Centre (commonly the centrosome or basal body), while the other is dynamic (the length can be changed if dimers are added or removed). Dynamic instability is, therefore, an attribute describing the ability of microtubules to extend through the process of polymerisation or retract through the process of depolymerisation.
Microtubules are polar structures, where the negative points towards the cell (nucleus) and positive points towards periphery of the cell.
Microtubules are critical for mitosis (by which cells replicate). During metaphase, microtubule fibres (mitotic spindle) hold on to the kinetochore in the chromosomes. This leads to the splitting of the chromosomes due to the shortening and thereby pull of the microtubules in anaphase.
Movement is another critical function provided by microtubules. Cilia (epithelium tissue) and flagella (sperm cell) are two different projections outside the cell that support movement. A protein called dynein breaks down ATP (adenosine triphosphate), using the energy to help the microtubules move past each other.
Finally, axonal transport in the neurons is a result of microtubules present inside nervous cells. Every neuron is made up of a cell body (soma) at one end and an axon (synaptic terminal) at other end. Kinesin (a motor protein) supports anterograde transport of items like neurotransmitters from the body to the synaptic terminal. Dynein (another motor protein) supports retrograde transport of items like old organelles from the axon back to the cell body.
Microfilaments
Formation of Microfilaments — monomer units called G-actin polymerise to form F-actin ; 2 polymer strands of F-actin twists into a double helix, resulting in the actin filament we recognise.
Similar to microtubules, microfilaments are polar, meaning they have a positive and a negative end. ATP helps new units join the positive end, while the hydrolysis of ATP into ADP causes units to disengage from the negative end. This assembly and disassembly to lengthen or shorten the microfilaments results in the filament’s movement.
Microfilaments can be a stimulating cause in the mitosis process (cell division). The final stage (cytokinesis), where division of the parent cell’s cytoplasm leads to the formation of two daughter cells. Here, microfilaments form a constriction ring which helps separate the two cells, allowing for cytokinesis to take place.
Cell extensions are achieved with the help of microfilaments. Cell membrane is divided into the apical surface and the basal surface, where apical faces the external environment and basal faces the basement of the cell. Extensions such as microvilli and stereo-cilia are formed at the apical section, helping improve functions like surface area.
Another key function of microfilaments in the cytoskeleton is membrane transport. Actin molecules can help support movement of items across the membrane (endocytosis and exocytosis).
Intermediate Filaments
Intermediate filaments come in different types, each serving different purposes based on their location in the body. For instance, desmond in muscle cells and fibroblasts in the connective tissue.
As compared to microtubules and microfilaments, intermediate filaments are not polar or dynamic, instead necessary for the structural support provided by the cytoskeleton. They resist mechanical stress and compressive forces, aiming to maintain cell shape.
DNA-Based Cytoskeleton
From the above understanding of the natural cytoskeleton, scientists were able to outline key characteristics required for a functional synthetic cytoskeleton. This included self- and reversible assembly, transporting ability and stability in the structure.
Different scientific groups and organisations have worked on carving a synthetic cytoskeleton based on DNA. Various theoretical concepts have been developed to imitate the natural workings of a cytoskeleton.
Reversible assembly
Strand displacement — DNA tiles are modified with toeholds, wherein invader and anti-invader strands can trigger assembly and disassembly of filaments. Invader strands are able to bind to the overhang of the DNA tile, leading to disassembly of the DNA tube. Anti-invader strands can bind to the invader strand, helping to reactivate the DNA tile and allow for reassembly. A study was done on this method’s effectiveness, and published in a research paper published in Nature. It found that the porosity (degree of filament assembly) fluctuated greatly in the presence of the invader and then anti-invader strands, indicating successful assembly and disassembly upon introduction of the strands.
Aptamer-target interactions — Another method used to instigate reversible assembly is aptamers. The DNA tile is prepared with a split aptamer that is present at opposite ends of the DNA tile. This creates a dual responsive filament, or a filament with the ability to respond and thereby reverse the processes of assembly and disassembly. Here, nucleolin (NCL) is used to trigger assembly, and adenosine triphosphate (ATP) for disassembly. When tested, the polymerisation process had key similarities between the DNA (synthetic) and actin (natural) cytoskeleton filaments, with both reaching similar degrees of polymerisation in 15 minutes.
pH — A triplex motif (triple-stranded DNA) has also been used in relation to Hoogsteen interactions for the assembly and disassembly of DNA nanotubes. Considering their pH sensitivity, Hoogsteen interactions become ideal for this use case, wherein a low pH leads to the motif binding to a complementary strand — this results in disassembly of the synthetic nanotube. However, this change is reversible, as an increase to a neutral or higher pH value leads to the triplex closing (instability of Hoogsteen interactions) — this brings about the reassembly of the DNA nanotube. The pH example outlines a process that is replicable with other cases, such as antigen DNA strands.
Intracellular Transport
RNase H — The cargo (item to be transported) is attached to the RNA overhangs in the DNA tile using a process called ‘complementary base-pairing’. This creates an RNA-DNA hybrid. Small Unilamellar Vesicles (SUVs) are prepared from DOPC lipids with the aim of mimicking natural transport vesicles found in cells. RNase H (ribonuclease H) is an enzyme that prompts hydrolysis, resulting in breaking the RNA strand in the RNA-DNA hybrid. The vesicle is no longer attached to the filament, instead binding with the RNA overhangs ahead (the binding is a result of its natural tendency). This process is referred to as the ‘Burnt Bridge’ method, as the RNA at the rear is depleted, leaving the vesicle with no choice but to move forward to RNA overhangs that have not yet been hydrolysed by the enzyme.
Electric fields — In this scenario, transport can be directed through rotational or translational motion. Rotational motion here would be in the form of a DNA arm made with origami. Translational motion, in this case, could be assumed using a tube DNA origami transporting system. While this is suitable considering DNA has a negative charge, the distance is limited by the length of the tube, and the speed of movement is slower to natural motors we see in real cytoskeletons.
Natural motors — Various natural motor proteins (e.g. dynein) can move DNA nanotubes. Motors can be engineered to DNA origami, helping improve the transportation speed. Taking the example of dynein, it binds and hydrolyses ATP, providing it with the energy to undergo propulsion. The cargo (item to be transported) is attached to the protein, allowing it to move along as well.
DNA-Peptide Crosslinkers
Peptide-DNA monomers consist of dipeptides that form fibrous structures and complementary DNA sequences. Changes in these two inter-linked parts lead to variation in the structures formed, with certain visible changing properties that make it easy to bring about variation in a synthetic cytoskeleton made of this material.
Temperature and DNA concentration as just two of many factors impacting the growth of these crosslinkers into a network, providing the insight that manipulation can effectively be rendered for structures of such materials.
The crosslinkers are able to mimic transport functions of a natural cytoskeleton. When polystyrene particles are introduced, some are trapped to confinements (similar to membranes in a natural cell). Other particles diffuse freely (Brownian motion), indicating the lack of interference of the DNA-peptide structure to movement.
Another key function of cytoskeletons are transient docking sites for elements such as proteins was discovered. Here, FITC-A (fluorescent marker) attached to a DNA strand became the payload. The FITC-A could bind to a complementary strand in the peptide-DNA network. When an invader DNA (A-I) was introduced, the FITC-A was displaced, leading to the release of the payload from the cytoskeleton. Confocal imaging helped us detect the reduction of fluorescence, indicating the clear release of the payload with the separation of the FITC-A from the skeleton.
Both these examples highlight the ability of peptide-DNA crosslinkers to replicate key functions of natural cytoskeletons, hinting at them being a viable solution to a synthetic cytoskeleton.
Microfluidics
Microfluidics, as the name suggests, works with fluids at very small scales, with a rough reference being a human hair (micrometers in diameter). Microfluidics can be involved in the development of a DNA cytoskeleton by mimicking the environment provided by a cell to the skeleton. The field allows for key components aside of the cytoskeleton to be encapsulated, along with easy testing of added elements such as vesicles, DNA strands, proteins and enzymes.
Many of the studies spoken about in this article involved the encapsulation of the filaments into cell-sized droplets, with the diameter engineer-able for different experiments. Mono-disperse droplets were formed, helping confine the filament structures created, improving testability of the DNA cytoskeleton. Sequential addition of molecules can be achieved using various microfluidic methods (fusion, pico-injection, light-triggered release, etc.).
Future of DNA Cytoskeletons
With DNA cytoskeletons presented and the development of peptide-DNA cross-linkers as a novel technology, it becomes necessary for us to work on the development of fully synthetic cells. Along with constant improvement in the creation of a cytoskeleton, the future holds developments in synthetic cells overall, where membranes, organelles and signalling pathways can be created for a fully functional cell unit.
The ‘Build-A-Cell’ initiative is an open community for individuals working in the field of synthetic cell development (building a cell ‘from scratch’). Along with many other initiatives, they aim to break the barrier to the synthetic cell, because no matter who breaks, there’s really no going back after. Similar to how the development of the first computer broke through a majority of the resistance, with a synthetic cell, it becomes a matter of modifying the genetic material as per the given use cases.
The global synthetic biology market is forecasted to grow to USD 35.7 billion by 2027, driven by the rise in demand for synthetic cells and reduction of DNA sequencing costs. The first synthetic cell in 2010 by the J Craig Venter Institute was the culmination of over $40 million over 15 years of research. However, this milestone step and the subsequent progress in the coming decade has led to a reduction of cost in the various applications.
For instance, the production of valuable materials by artificial cells has led to, for instance, two to five times the cost of regular plastic production. While this may sound expensive, with the average cost of regular plastic production per kilogram being between $2 and $5, the production of 1 kilogram of synthetic cells in certain uses is as low as $5! This clearly reflects major progress in the costs of production of synthetic cells, making them more and more integrable into existing products, both now and in the future.
However, synthetic cells are not solely limited to the production of materials. When we look at the biomedical field, one of the most booming industries when it comes to innovation, synthetic cells are one of the largest projected markets. For instance, the synthetic stem cells market alone is predicted to grow to USD 104.88 million by 2032, reflecting a compound annual growth rate (CAGR) of nearly 23%! And considering synthetic cells are the precursor to every specialised cell in our body, this will result in impacts on every domain of healthcare, from the cardiovascular to the neurological.
When we look at the reliability of its different uses, synthetic cells are yet to reach required levels of accuracy for implementation in real patient treatments.
- Treating hypercholesterolemia by inducing a mutation in the PCSK9 gene. The use of two synthetically engineered cells resulted in 54% and 56.5% of successful mutations accordingly.
- The treatment of Type 1 Diabetes with the use of stem-cells differentiated into insulin-producing pancreatic Beta-cells (engineered in the lab). Here, one specific therapy (Vertex’s VX-880) led to a reported 91% decrease in daily insulin requirements.
- The reduction in toxicity of norcantharidin (NCTD), an anticancer agent, with the use of nano-technology based systems. Various systems were used, yielding different tumour inhibition rates : liposome systems resulted in a 48.4% difference, integrated micelle systems a 77.63% as compared to 47.5% with free NCTD, and NCTD nanoparticles a 48.4% rate in contrast to 37.6% with free NCTD.
These are just a few of countless application examples for synthetic cells. Truly, if you can think of any real-world scenario, there is a potential living being that can be synthetically engineered. When we think of the world’s biggest problems : resource scarcity, climate change, diseases and viruses, global waste systems, there are theoretical synthetic solutions waiting to be applied.
We are so close to finding the key to engineering life — it’s no longer a question of if, but when. What we need to be asking ourselves is : what doors can this key unlock?