Future Directions

PART1: Biotechnological mass production

DNA origami is a nanostructure assembled from a very long single-stranded scaffold molecule, which is held in place by many short staple oligonucleotides. Bacteriophage-derived scaffold molecules can be feasibly produced at scale, while the shorter staple strands are typically obtained through costly solid-phase synthesis or enzymatic processes (1). Fortunately, an innovative method for the phage-free production of short single-strand DNA staples using E. coli has been developed in recent years. This enables the biotechnological production of complete DNA origami without cross-contamination. To assess the affordability of our therapeutic for the general population, we plan to partner with the research and development division of a local pharmaceutical factory to construct a test model.

Design Criterion

The engineered E. coli system consists of an artificial phagemid and a helper plasmid. The former contains the crucial regulatory sequences required to initiate rolling circle amplification—a specific amplification pattern derived from phages that produces long single-strand DNA—but it lacks the genes for capsid proteins. The latter incorporates both the phage backbone and a regular plasmid backbone, enabling the continuous production of the main capsid protein but not the initiation of rolling circle amplification. This design prevents the phages from self-replicating, thereby eliminating the risk of engineered phage residues being released into the environment. Simultaneously, the separation and controllability of these components enhance production efficiency.

Phagemid and helper plasmid design
Figure 1. An example of phagemid and helper plasmid with crucial components, taken from reference (2)

Detailed Plasmid Design Concept

In our design, the crucial sequence for initiating rolling circle amplification—F1-ori (2)—is regulated by a promoter that is only active when stimulated by specific substances. Otherwise, the engineered bacteria will maintain normal double-strand DNA replication to accumulate capsid proteins and DNA material until a critical density is reached.

The plasmid's main component is an artificial pseudogene, into which all target staple sequences (including m13) are interleaved with self-cleaving catalytic DNAzymes. Each DNAzyme is approximately 70 base pairs long. Due to time constraints, we use one type of staples (F-cap-x) as an example and design its corresponding pseudogene sequence.

Design criteria illustration
Figure 2. Illustration of the design criteria and effect of user-defined sequences, which will be cleaved into scaffold and staples of DNA origami. Figure taken from reference (1).

The sequences

View sequence details (PDF)

Industrial Flow chart

Industrial production flowchart
Figure 3. Illustration of the procedure for biologically producing DNA origami. Engineered E. coli is first cultured until bacterial accumulation reaches a critical density. If not, cultivation continues; if yes, a signaling substance is added to initiate rolling circle amplification so that phages unable to replicate themselves are produced. Then, phages are collected and subjected to DNA purification. After adding Zn²⁺ to facilitate DNA self-excision to obtain scaffold and staple strands, a PCR program is run to construct the DNA origami, which is then stored before transported applying to the patients.

Rough Calculation of Production Costs

With biotechnological mass production, the cost is estimated to be approximately €0.18 per milligram of folded DNA origami in an 800-liter volume at a contract biotech facility. This is at least three orders of magnitude cheaper than the estimated cost of producing the same amount via conventional solid-phase chemical oligonucleotide synthesis. Furthermore, costs decrease with an expansion of the production scale (1).

Cost breakdown
Figure 4. Cost breakdown for DNA origami nanorod production at lab scale and pilot scale, adapted from reference (1)

PART 2: Expansion of Implementation Scenarios

From Wound to Respiratory and Intracranial Infections

In our initial design, the drug delivery system is encapsulated within a hydrogel and applied to or sprayed on skin wounds infected by Methicillin-resistant Staphylococcus aureus (MRSA), a common occurrence in hospital ICUs where patients are frequently exposed to antimicrobial-resistant bacteria. However, the delivery strategy must be expanded to address more severe and advanced-stage infections, such as those in the respiratory tract and intracranial region (3). Our therapeutic, Focas, is an efficient and biocompatible platform that can be readily adapted to other delivery methods to treat a broader range of diseases. Here, we present a conceptual framework for future designs in these two areas, based on existing research.

Respiratory Infections

MRSA respiratory infections can cause life-threatening diseases, including pneumonia, empyema, and respiratory failure. Conventional oral and parenteral antibiotic treatments are often inefficient at targeting the lower respiratory tract and can cause systemic adverse effects (4). In contrast, nebulized inhalation is considered an optimal method for lung disease treatment due to the long drug retention time in the lungs, improved delivery of hydrophobic drugs, and reduced systemic toxicity. Specifically, nebulized samples are administered as 10–50 μm microdroplets via a nebulizer. Focas possesses the characteristics of nanomedicines that are likely to pass through the airways and cross the mucus barrier. Previous studies have shown that DNA origami-based drugs can successfully diffuse into deeper regions and remain stable. We plan to conduct experiments to validate our design. The potential crucial role of peptide modification in the treatment requires further evidence (5).

Nebulized drug delivery
Figure 5. Schematic diagram of nebulized drug delivery of Focas in new implementation scenarios.

Intracranial Infections

Bacterial meningitis is a severe infection associated with a high mortality risk. A study in Ontario, Canada, showed that Staphylococcus aureus was the second most common pathogen in community-acquired meningitis and the most common pathogen in nosocomial meningitis (6). The ability of bacteria to cross the blood-brain barrier (BBB) and proliferate slowly in the subarachnoid space presents a significant treatment challenge (7). Our adjusted Focas platform functions as a composite delivery vesicle designed to transport CRISPR-Cas9 together with traditional antimicrobial small molecules across the blood-brain barrier. This system is designed to simultaneously kill bacteria and prevent the spread of antimicrobial resistance genes. The barrel structure of Focas has been shown to cross the BBB via transcytosis more efficiently than smaller, rigid structures. In the next phase, we will modify the scaffold into a soccer ball configuration, which offers greater flexibility and deformability, and test its transport efficiency (8).

DNA origami morphologies
Figure 6. Different origami morphologies and their transport efficiency through BBB, figure taken from reference (8).

PART 3: Stability Enhancement

Although our wet lab experiments indicate that Focas can maintain its structure for a few hours in PBS and a few days when stored at 4°C. However, the therapeutic must remain stable long enough to be effective in a more complex body environment when delivered through vascular system or pulmonary mucosa. Moreover, DNA origami material should be able to store for much longer time in practical implementation. Therefore, our future plan is to enhance stability of Focas by analyzing both exogenous and endogenous factors.

Exogenous Reasons and Enhancement Plan

In the complex wound microenvironment, the degradation of free DNase enzymes and lysosomal in host cells like phagocytes poses a threat to the stability of the entire biomaterial delivery platform. A potential solution involves using a bio-reducible, cationic polymer—poly(cystaminebisacrylamide-1,6-diaminohexane) (PCD)—as a reversible protective capsule for the DNA origami. Previous studies have demonstrated PCD's protective effect against low salt conditions and DNase I degradation. Interestingly, it may also enhance target combination (9). We will test the stability of Focas encapsulated with PCD in a modeled wound microenvironment and concurrently collect data on changes to aptamer targeting efficiency.

Endogenous Reasons and Enhancement Plan

The membrane rupture component of Focas is G4-hemin, which generates reactive oxidative species (ROS) to disrupt the bacterial outer membrane and facilitate drug internalization. Although our wet lab shows Focas can withstand attack from ROS in experiment time, we still doubt it causing unnecessary injury on DNA origami structure in a longer time period. To replace this critical membrane transport component, we have identified potential in targeting cell surface thiols to enhance cellular uptake. Related strategies involving disulfide conjugation to nanoparticles, oligonucleotides, and even DNA origami have been proven as an efficient and rapid strategy with no associated toxicity (10-12). However, direct evidence in prokaryotes is lacking due to limited research. We plan to collaborate with laboratories specializing in prokaryotic transmembrane drug delivery to test the feasibility of this new method. If the results are positive, we will replace the G4-hemin component with staples modified with disulfide units on the 3' terminus and conduct subsequent confirmatory experiments.

Staples modified with disulfide units
Figure 7. Staples modified with disulfide units to enhance cellular uptake, taking SiRNA drug as an example, taken from reference (11).

Remaining Problems and Future Prospects

As a versatile bacterial-killing platform, Focas still holds great potential for future iGEM teams to optimize. For instance, the lock-staples containing chemically synthesized disulfide bonds present a barrier to mass production due to their high cost. Is there a biological alternative to produce this type of specialized staple, or another mechanism for CRISPR cellular escape in prokaryotes? Furthermore, a standardized model for loading traditional antibiotic molecules onto Focas needs to be established to simultaneously kill bacteria and prevent the spread of drug resistance genes.

In summary, although Focas still has a long development path ahead before clinical implementation, we believe it represents the beginning of a major advancement. We are confident in pioneering a standardized, quantitative, and universal biopharmaceutical platform targeting drug-resistant bacteria, and we hope to provide a foundation for future iGEM teams to build upon.

References

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  2. Behler KL, Honemann MN, Silva-Santos AR, Dietz H, Weuster-Botz D. Phage-free production of artificial ssDNA with Escherichia coli. Biotechnology and Bioengineering [Internet]. 2022 [cited 2025 Sept 11];119(10):2878–89. Available from: https://onlinelibrary.wiley.com/doi/abs/10.1002/bit.28171
  3. Nandhini P, Kumar P, Mickymaray S, Alothaim AS, Somasundaram J, Rajan M. Recent Developments in Methicillin-Resistant Staphylococcus aureus (MRSA) Treatment: A Review. Antibiotics (Basel) [Internet]. 2022 Apr 29 [cited 2025 Sept 12];11(5):606. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9137690/
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  6. Bai AD, Boyd JG, Li W, Campbell RJ, Gill SS. Microbiology and long-term outcomes in community-acquired, nosocomial and post-neurosurgical meningitis with positive bacterial culture in Ontario, Canada: A population-based cohort study International Journal of Infectious Diseases [Internet]. 2025 Aug 1 [cited 2025 Sept 12];157:107925. Available from: https://www.sciencedirect.com/science/article/pii/S1201971225001493
  7. Sharma H, Narayanan KB, Ghosh S, Singh KK, Rehan P, Amist AD, et al. Nanotherapeutics for Meningitis: Enhancing Drug Delivery Across the Blood-Brain Barrier. Biomimetics (Basel) [Internet]. 2025 Jan 3 [cited 2025 Sept 12];10(1):25. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11762342/
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  9. Youssef S, Tsang E, Samanta A, Kumar V, Gothelf KV. Reversible Protection and Targeted Delivery of DNA Origami with a Disulfide-Containing Cationic Polymer. Small [Internet]. 2024 [cited 2025 Sept 13];20(10):2301058. Available from: https://onlinelibrary.wiley.com/doi/abs/10.1002/smll.202301058
  10. Torres AG, Gait MJ. Exploiting cell surface thiols to enhance cellular uptake Trends in Biotechnology [Internet]. 2012 Apr 1 [cited 2025 Sept 13];30(4):185–90. Available from: https://www.sciencedirect.com/science/article/pii/S016777991100206X
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