LOW CARBON ECONOMY
The Importance of Nanorobotics
Once deemed fantasies created by science fiction, the field of nanorobotics has expanded immensely. Introduced in 1986, K. Eric Drexler’s book allowed for the creation of a multi-billion dollar industry . Today, most DNA nanotechnology utilizes a technique known as DNA origami, a procedure that takes advantage of the intrinsic self-assembling capabilities of DNA to construct complex nanoscale structures . This was accomplished through the mixing of short strands of DNA with specific sequences such that when mixed with a long single stranded DNA ‘scaffold’, the short strands ‘staple’ the scaffold at precise locations to fold the structure into a predetermined confirmation . The near limitless potential confirmations with which the DNA can be folded created doors for diverse functionalization .
The First Steps Toward Actuation
Since the genesis of this new field, the latest advances have resulted in structures capable of executing basic tasks, such as opening and closing, functionally mimicking simple macroscale machines. In January 2015, a group of mechanical engineers from Ohio State University decided to create nanorobots using DNA origami approaches and basic design principles as shown in figure 1. .
First, a DNA hinge was created using two stiff arms that allow for purely constrained rotations. Furthermore, these two stiff arms were joined along an edge by flexible ssDNA (single-stranded DNA) scaffolds which allowed for rotation within 90० degrees. The team then built a piston and cylinder system using three hinges, three planks and two tubes of different diameters. These applications of mechanical systems created the first actuation of DNA but presented several issues.
Figure 1. Typical machine parts juxtaposed with their dsDNA origami counterparts .
Why Improvements Were Needed
While the developments made by the team represented a major leap forward, the primary limitation of their structures comes from an inability to control them in a timely manner. Reducing these long latencies to sub-second timescales presents many future applications that could be utilized in more complex tasks that require both precise and quick actuation. This lays the foundation for a future in more advanced cellular work such as nanomedicine .
Real-Time Actuation And Where UBC BIOMOD Comes In
Developing a new mechanism of actuation of these structures, in conjunction with polymerization of these hinges, allows for modular control. The criterion for this new mechanism would be the following:
- The mechanism is modular (can be controlled and fine-tuned at certain angles) without an external field.
- The mechanism is cheap and cost-effective.
- The mechanism contributes to the low-carbon economy (which follows the green principles of chemistry ) through low use of carbon in both the mechanism and the manufacturing process. (see Figure 2)
The advancements made by Ohio State University have brought the field of nanotechnology one step closer to more advanced applications. However, expanding upon these advancements is scientifically and financially is a challenge. The nanohinge presented in the Ohio State paper allowed for rotation about a fixed point and used an external magnetic field for actuation. UBC BIOMOD aimed to expand upon this by creating a system of repeating monomer hinges, allowing for actuation across a distance, rather than an angle. Finally, UBC BIOMOD created a unique mechanism that would allow for modular, precise, self-actuation of the hinge. This advancement will pave the way for more complex applications such as biomedical application and filtration.
Figure 2. The twelve green principles of chemistry. Green chemistry is used to truly eliminate environmental contaminants by gauging the manufacturing process  from beginning to the end. Reproduced without permission from [Hems et. al, 2021].
A problem with designing bigger and more complex DNA origami structures is that these structure require more staple strands to fold the scaffold into the correct shape. However, as the number of staples increases the probability of sequence overlap increases as well. This is problematic as the incorrect binding of staple strands to the scaffold may result in improper structure folding.
In response, we designed a DNA nanohinge system that can be scaled and reversibly locked into different conformations. To accomplish this, we designed a pair of hinges that can polymerize arm to arm, to create a larger structure consisting of repeatable units. These hinges are denoted as H1 and H2.
H1 and H2 are both comprised of 2 nanobricks. Each nanobrick comprises 56 double-stranded DNA helices connected in a honeycomb lattice conformation. A single nanobrick is expected to be approximately 50 nm in length, 15 nm in height, and 25 nm in width. Nanobricks are held together by short oligonucleotides, known as staple strands, that are 15 to 60 bp long. Each staple strand has a predetermined location on the scaffold to which it is expected to bind based on sequence complementarity. The hybridization of the staple strands to the scaffold backbone forces the scaffold to adopt a rectangular 3D shape. Two separate bricks are both made on the same scaffold, allowing the structure to adopt an overall hinge-like shape, the nanohinge.
Figure 3. Structure scaling
H1 and H2 are represented as the in blue and red hinge in Figure 3 respectively. The end of the bottom brick of H1 can only be attached to the end of the top brick of H2. The same specificity goes for the alternative bricks. This is accomplished through the incorporation of polymerization strands. Each polymerization strand has a complementary region to the H1 scaffold and the H2 scaffold. This allows for H1 and H2 to self-polymerize into long chains when incubated in the presence of the polymerization strands. This design approach reduces the number of different staple strands needed to form a larger and more complex structure.
We refer to structures consisting of more than one hinge unit as an extended hinge structure (EHS). EHSs are able to expand and contract along a linear axis, similar to the motion of an accordion. This motion is accomplished through the incorporation of the locking mechanism described below.
We designed a DNA-based reversible locking mechanism that allows for each hinge to be an open or closed conformation. The structure can be ‘locked’ or ‘unlocked’ using toe-hold mediated DNA strand displacement. The ability to ‘lock’ the structure in certain configurations allows for the structure to have interesting applications which are discussed below. Starting with the hinge in a closed position, this conformation is maintained by two complementary ssDNA linker strands termed a “lock strand” and a “padlock strand”. The smaller lock strand keeps the padlock strand in place allowing for a rigid closed hinge position. To put the hinge in an open position, a longer ssDNA strand complementary to the padlock strand called the “key strand” displaces the lock strand. Afterward, an anti-lock strand binds with the lock strand to prevent unnecessary structure binding allowing the hinge to remain open. The key strand then gets displaced by a complementary sequence called “the anti-key strand” to free the padlock strand and is replaced by a new lock strand.
Figure 4. Locking mechanism schematic
The molecular diagnostics market plays a key role in the rapid detection and control of new diseases, as recently illustrated by the role of reverse transcription-polymerase chain reaction (RT-PCR) in the course of the SARS-CoV-2 pandemic. Challenges facing the use of such methods of diagnostic such as chromatography, immunoassays, and sequencing include low throughput and sensitivity, high assay and equipment maintenance costs, and the need for trained personnel , . In the context of existing inequities in access to healthcare facilities, these factors can serve to exacerbate discrepancies in healthcare outcomes between regions. In an acute sense, these interactions can lead to significant barriers in public health situations requiring high-throughput screening, also illustrated in the recent pandemic. Our project addresses the two overall aspects of technical innovation (sensitive, configurable diagnostic tools).
Using the fact that the hinge has two conformations, open and closed, a quencher-fluorophore system can be incorporated into the locking mechanism for diagnostics purposes. The biomarker would be the ‘key’, displacing the locking strand, which results in the closed to open conformation. This results in the emission of a measurable signal from a fluorophore affixed to one hinge arm as it distances from a quencher attached to the other arm(results of this experiment will be posted after). The hinges can thus be configured as highly sensitive biosensors for nucleic-acid based biomarkers. Hinge and strand solutions can be lyophilized and re-suspended in various dilutions on-site for rapid diagnostic use, requiring only a plate reader and plastic well plates. A library of locking strands with regions specific to different nucleic acid biomarkers can be used for the detection of different pathogens. Each specific locking strand sequence would only open the hinge upon hybridization to the targeted biomarker, significantly lowering the chance of false detection, and allowing for rapid visual evaluation of pathogen presence.
Utilizing the cyclic nature of the locking mechanism, we propose a reusable nucleic acid sequence detection assay. Difference in fluorescence from the fluorophore-quencher system in open and closed confirmation can be detected using a spectrophotometer. A padlock strand can be designed to a complementary to the nucleic acid seuqnce of interest to detect for the existence of this sequence, such as in the viral genome of COVID 19. The detection of presence of sequence, therefore virus, using a spectrophotometer would be relatively faster than PCR or qPCR assays and it would produce less plastic waste, as the assay would be reusable (although it will need to be validated between each assay).
The structural design of our project is split into 5 main cadnano-based components of which we would focus on creating, completing and optimizing within the months given, allocated to design, validation, then construction. However, the bottleneck of the project revolves around the creation and confirmation of the initial caDNAno and CanDo designs as a foundation, prolonging the project for many months. Confirmation of individual components is assigned after their completion, which would be followed by further confirmation of attachment and function upon assembly. This would go in the order from designing and producing the individual DNA origami components (nanobricks, nano hinge, cap structure), incorporating the paramagnetic bead, and the locking strands.
Goal 1: Hinge Design
- Design two DNA nanostructure hinges that can connect end to end in an alternating pattern
- Design lock and key mechanism to fix hinges in certain conformations
Goal 2: Hinge Assembly
- Confirm the assembly of each individual DNA hinge unit
- Confirm the ability of the single hinge units to to hybridize and bind to each other and form an extended hinge structure
Goal 3: Actuation of Hinges
1. To demonstrate the ability of the lock and key mechanism to reversibly fix the DNA hinges in specific conformations
LOW CARBON ECONOMY
Single-use, point-of-care diagnostic tests are important for responding to urgent medical crises, especially where laboratory facilities and resources may be limited. However, they pose a significant environmental challenge. Rather than sustainability, point-of-care diagnostic test manufacturing is often driven by compatibility with current infrastructure and economic benefit (11). After manufacturing, carbon emissions from long transportation distances and disposal by incineration contribute to their environmental burden. The COVID-19 pandemic has highlighted the importance of diagnostic tests, but also exposed the current flaws in their unsustainable design and lack of worldwide access (12–14). For example, until August 2020, over 15,000 tons of COVID-19 polymerase chain reaction (PCR) test plastic waste had been produced (13). The two primary disposal routes for COVID-19 PCR plastic waste are by incineration, where toxic organic pollutants may be released into the atmosphere (15), or in a landfill, where harmful microplastics can spread into the surrounding environment (12,13).
To alleviate this impact, we are proposing the development of a DNA origami-based structure that could be used in multi-use, multi-purpose diagnostic devices. We envision our structure to have potential use in diagnostic applications in the following manner: DNA nano-hinges can be biotinylated to a streptavidin-coated surface in a closed conformation with aptamer-controlled actuation at each nano-hinges’ free end. Upon antigen-aptamer binding, a nano-hinge will change to an open conformation, causing the release of a quencher-fluorophore interaction and subsequent fluorescence emission detected by the device. The device can be reused by eluting bound molecules from the detection surface and sterilizing it. The possibility for re-use and simultaneous detection of multiple antigens will decrease the number of tests transported to areas in need, thus decreasing transportation-based carbon emissions. In addition, our device could be repurposed by either creating extended hinge systems on the detection surface with different aptamer controlled hinge actuation, or by removal of the first layer of hinge and addition of a new layer. Plastics under ambient solar radiation in landfills can produce carbon emissions that have yet to be well characterized (16). A reusable device could reduce the amount of single-use plastic waste put in landfills and their carbon emissions under solar radiation. Reducing waste is especially important during a medical crisis, such as the COVID-19 pandemic, where an immediate influx of waste can disrupt existing disposal protocols and exacerbate current waste management issues. Overall, we aim to decrease the future carbon footprint of COVID-19 and other diagnostic tests with a DNA origami-based device.
The filtration of particles through vessels and passageways. The USYD 2019 Biomod project of LDL filtration is a good example of this. The gaps in the structure could be used to allow through particles below a certain size. The “pore” size would be controlled by magnetic actuation within a certain range, subject to the dimensions of the components, which can also be controlled in their synthesis. 3-D filter arrays would ensure the exclusion of larger particles. Larger molecules could be filtered for through 3-D arrays effectively altering the volume of passageways and vessels.
One of the applications mentioned would be to use superparamagnetic beads (tiny magnets) to the polymerized hinge. This would require the use of biotin overhangs attached using the process of biotinylation. Essentially, biotinylation, used as an attachment system, involves conjugating biotin molecules to ssDNA ‘overhangs’ and then taking advantage of biotin’s strong affinity for streptavidin which is coated on the surface of a coverslip.  Through this interaction, the nanostructures become strongly anchored to the coverslip. An external magnetic field would then be applied to actuate the nanohinge in a more precise manner (i.e. able to take continuous angles instead of discrete).
The selective capture of certain structures and particles. Buckminsterfullerene cages can be roughly seen as a downsized example. This purpose would entail chemical or structural alteration of the mesh to target a particular morphology of bacteria, for example, or specific structural elements of distinct chemical affinities and either degrade and alter the captured structures in some way or sequester them by retraction for manual removal.
Altering the shape and volume of structures such as cells and organelles. This can be done by the addition of effector molecules, to repulse and attract, onto the arms ends of mesh structures affixed within the target zone and can be considered an extension of force application.
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