DNA is universally recognized as the primary genetic material that transmits hereditary information in humans and nearly all other organisms. As the genome, it serves as the blueprint that governs cellular functions, encoded in a complex molecular language. Since Watson and Crick's groundbreaking discovery of its double helix structure in 1953, extensive research has revealed the remarkable potential of this molecule. DNA's exceptional programmability is driven by its complementary base pairing (A-T and G-C), which forms a stable, spiral staircase-like duplex. This inherent programmability, combined with its precise base-pairing, robust physicochemical stability, and the ability to be synthesized autonomously, makes DNA an ideal platform for constructing custom molecular structures. (1)
Over the past four decades, research has revealed that DNA is not only a genetic molecule but also a powerful material for constructing nanoscale structures with remarkable precision. Prior to the 1980s, the idea of utilizing DNA as a scaffold for assembling guest molecules into nanoscale objects was largely unrecognized. This innovative concept was first proposed in 1982 by Nadrian Seeman, a pioneer in the field and the founder of DNA nanotechnology. (2-4)
DNA nanotechnology involves the design and fabrication of artificial DNA structures for technological applications, utilizing DNA as a non-biological engineering material. Inspired by M.C. Escher’s woodcut ‘Depth’, which resembles the intricate patterns of six-arm DNA junctions, Nadrian Seeman recognized the potential to create 3D lattices with DNA that could serve multiple functions. In 1991, his laboratory successfully synthesized the first three-dimensional nanoscale object—a DNA cube. 5 Later, in 1999, Seeman’s team demonstrated the first DNA nanomachine, a device capable of altering its structure in response to external inputs.(6)
The concept of creating stationary DNA nanostructures was inspired by the four-arm mobile Holliday junctions found in DNA, which play a critical role in meiotic cell division. 7 This structure is topologically similar to a crossroad junction, where two roads intersect perpendicularly, creating four arms that move in opposite directions. The point where the strands exchange, known as the branch point or junction, is essential for recombination between homologous DNA duplexes, contributing to genetic diversity. By disrupting the sequence symmetry of the Holliday intermediate, researchers stabilized the previously mobile four-arm junction, using it as a foundational unit for constructing more complex, rigid structures.
DNA is inherently biocompatible, biodegradable, minimally toxic, and versatile in its biological functions. Thanks to advancements in synthesis and bioconjugation techniques, DNA strands can be easily modified to incorporate various functional groups, such as macromolecule carriers or targeting ligands, thereby enhancing the functionality of delivery systems. Over the past four decades, significant progress in DNA molecular self-assembly (Figure 1) has led to the development of precisely engineered DNA nanostructures, which have shown great promise as drug delivery vehicles, demonstrating high delivery efficiency in both in vitro and in vivo applications.(8-9)
In this cover story, the development of DNA nanostructures and their emerging role as drug delivery vehicles for the targeted transport of both small molecules and macromolecular drugs have been explored. Additionally, the potential benefits and challenges associated with utilizing these nanoparticles in drug delivery applications have been highlighted.
Figure 1: Self-assembly of DNA molecules creating DNA objects for targeted drug delivery
Methods of Developing DNA Nanostructures
Molecular self-assembly plays a fundamental role in the structural diversity and complex functionality of biological systems with information-carrying molecules such as DNA. For therapeutic applications, the fabrication of complex DNA nanostructures mostly relies on the self-assembly of DNA strands. To this end, DNA as a building block, plays a dual role as a biomaterial and a glue for spontaneous assembly. Since the original proposal of Nadrian Seeman, a plethora of approaches and methodologies have been tried with the self-assembly of DNA nanostructures in the past four decades. Most of the techniques were based on the sticky-ended cohesion between the complementary strands of the DNA molecules. Analogous to hook & loop flaps of Velcro, sticky ends are short single-stranded overhangs originating from the ends of a double-stranded DNA helix that have complementary arrangements of the nucleotide bases (A-T, G-C) to form complex molecular structures. (2) Utilizing this unique Watson-Crick base paring principle, a variety of DNA nanostructures were created by hybridization of complementary single-stranded extensions or sticky ends.
DNA nanotechnology is a unique field, where physics, chemistry, biology, mathematics, engineering, and materials science can elegantly converge for creating useful objects with nanometer precision. The design of DNA nanostructure plays a pivotal role that in many cases also dictates its application(s). In its very childhood of DNA nanotechnology, Seeman’s group introduced a tile-based method for DNA assembly where bundled DNA strands resulted from DNA crossovers form a variety of units called ‘tiles’. Repeated tiles were interconnected by complementary single-stranded sticky ends to form larger objects. Tile-based approach opened the door for the construction of highly ordered 1D, 2D, and 3D DNA structures. Over the last four decades, the library of DNA nanostructures has been enriched with geometric shapes, polyhedral objects, smiling faces, nanoscale bunnies, a box with a lid and even a miniature Mona Lisa. (10 )
After nearly 25 years of dedicated research, creating larger DNA nanostructures remains a significant challenge. In 2006, Paul Rothemund at the California Institute of Technology addressed this gap by developing the DNA Origami technique, which simplified the complex task of constructing larger DNA structures and propelled the field forward with remarkable momentum.(11) The Origami technique draws inspiration from traditional paper folding, where a long single-stranded DNA molecule acts as a scaffold and is folded into 2D or 3D shapes using hundreds of short complementary ‘staple’ strands. Unlike earlier tile-based methods, the origami approach allows for the creation of much larger and more intricate structures, expanding the possibilities for DNA nanotechnology.
Seeman's laboratory achieved a major milestone in DNA nanotechnology by developing the first rationally designed 3D DNA crystals using tensegrity triangular tiles.12 This groundbreaking work marked a significant advancement in the field. Table 1 provides a summary of the key milestones and progress made in DNA nanotechnology over the past four decades.
Table1: Landmark Events in DNA Nanotechnology
Targeted therapy is an approach that uses therapeutic agent(s) to attack specific types of cancer cells without causing any significant damage to the normal cells. A big challenge in nanomedicine is to design suitable nanocarriers that can precisely transport therapeutic agents to the target site in the complex environment of living organisms. Conventional anti-tumor drugs often suffer from shortcomings, such as targeting multiple points, high cellular toxicity, and poor in vivo stability leading to poor therapeutic outcomes. (18) In recent years, DNA nanostructures have garnered significant attention in biomedicine for their potential as drug delivery carriers. These structures take advantage of DNA's unique properties to create highly programmable and versatile platforms capable of delivering both small molecules and macromolecular therapeutic agents. One of DNA's inherent strengths, in contrast to other materials, is its ability to perform molecular computation, which has been harnessed to program DNA nanostructures for in vivo drug delivery. Additionally, DNA offers unparalleled control over the size and geometry of nanostructures, enabling the creation of homogeneous drug delivery platforms that distinguish them from conventional self-assembling nanoparticles.
DNA nanostructures can be tailored to carry a wide range of complex drug cargos, including monoclonal antibodies for immunotherapy, small interfering RNAs (siRNAs) and antisense oligonucleotides for gene therapy, and proteins for vaccine development. For instance, Harvard Medical School, in collaboration with Korea Institute of Science and Technology, developed an origami-based vaccine, DoriVac,(19) which demonstrated exceptional stimulation of antigen-presenting cells (APCs), leading to a highly favorable T-cell response. DoriVac is a self-assembled square block (SQB) nanostructure incorporating cytosine-phosphate-guanine (CpG) adjuvant molecule, a synthetic DNA strand that contains repeated CpG motifs. These motifs mimic genetic material from bacterial and viral pathogens, triggering Toll-like receptors in macrophages and dendritic cells, which in turn activate T-cells. DoriVac also exhibited synergistic inhibitory effects alongside immune checkpoint therapies, which have already been successfully used in clinical immunotherapy.
DNA nanostructures can also serve as carriers for genes or gene-editing tools, such as CRISPR/Cas9. Gene-editing is a technique to precisely prune, cut, replace, or insert DNA or RNA sequences for therapeutic purposes. As efficient delivery of CRISPR/Cas9 to target cells is a considerable challenge, Sun et. al. developed a new type of self-assembled DNA nanostructure through rolling circle amplification (RCA), which can be used to deliver CRISPR/Cas9 RNP in vitro and in vivo. Table 2 provides a summary of selected DNA nanocarriers used in the delivery of various therapeutic and diagnostic agents.
Table 2: Selected DNA Nanocarriers Used in Drug Delivery/Diagnostics
DNA nanostructures offer several significant advantages over traditional drug delivery carriers, particularly in targeted cancer therapy. The programmability of DNA allows for easy modifications of nanostructures, enabling customization of their size, shape, and surface chemistry to optimize drug delivery. (33) DNA can be engineered to form specific shapes, such as nanotubes, cages, or dendrimers, and programmed to release their drug payloads in response to precise stimuli, such as changes in pH, temperature, or the presence of specific enzymes. This ability ensures that drugs are released in a controlled manner, specifically at the tumor site. A generalized approach of drug delivery with DNA nanocarrier and its release mechanism has been shown in Figure 2.
Figure 2: DNA nanostructures used as drug delivery carriers to specific sites in the body. DNA origami nanotube loaded with drug is targeted towards tumors using a nucleolin-targeting aptamer. On reaching the tumor, the nanotube is opened by interaction of the aptamer with the protein nucleolin. (Used with permission from the author of Ref. 10)
In addition, DNA is inherently biocompatible and biodegradable, minimizing the risk of adverse reactions within the body. DNA nanostructures can be tailored to recognize specific cancer cell markers, enabling targeted delivery to tumor cells while sparing healthy tissues. Their nanoscale size enhances their ability to penetrate tumor cells and tissues, improving the therapeutic efficacy of the drugs.(34) Furthermore, DNA nanostructures can be tagged with imaging agents, allowing for real-time monitoring of their distribution and drug release in vivo.
DNA nanostructures are particularly well-suited for delivering nucleic acid-based therapeutics, given their chemical similarity to DNA and RNA.(35) They have already demonstrated success in delivering macromolecular drugs, such as microRNAs, small interfering RNAs, and antisense oligonucleotides. Another exciting area of research is the incorporation of protein components into DNA nanostructures, opening new possibilities for the development of vaccines, biosensors, and enzyme replacement therapies.
The unique advantages of using DNA nanostructures as drug delivery vehicles stem from their intrinsic biocompatibility, high programmability, robust self-assembly, and effective drug loading capabilities, among others. Additionally, DNA structures can be structurally modified to incorporate other molecules, such as gold nanoparticles, further enhancing their functionality. With the added benefits of biocompatibility, rapid renal clearance, and minimal toxicity, functionally modified DNA nanostructures excel in controlling systemic circulation time, drug release kinetics, and target-site specificity. These characteristics significantly improve the efficacy of encapsulated drugs, making them highly efficient for targeted therapeutic applications. (10)
Challenges Towards Translational Applications
DNA nanotechnology holds immense promise across various fields due to its ability to create complex, precisely engineered nanostructures using DNA as a building block. However, it faces several challenges when it comes to transitioning from the laboratory to real-world applications.
While DNA itself is structurally stable, with an approximate half-life of 500 years,(36) DNA-based nanostructures can be compromised by environmental factors such as enzymes, pH variations, temperature fluctuations, and ionic concentrations. In particular, DNA nanostructures are highly vulnerable to nucleases present in body fluids like blood, urine, and saliva. Although nucleases play critical biological roles, such as in topoisomerization, site-specific recombination, and RNA splicing, they also degrade DNA-based structures, interfering with their intended functions in biological systems. As a result, stabilizing DNA nanostructures in these hostile conditions while maintaining their biomedical functions remains one of the primary challenges for their clinical translation.
Another significant hurdle is the large-scale manufacturing of DNA nanostructures that meet the quality and reproducibility standards required for clinical use. While small-scale synthesis and structural modifications are relatively straightforward, scaling up production of DNA nanostructures can be costly, limiting their accessibility and viability as therapeutic options. Moreover, for therapeutic DNA nanostructures to enter clinical trials, production cost is another concern, especially when chemical modifications are in high demand.(37) Therefore, the cost of DNA nanomaterial synthesis should not be underestimated. Because complex nanocarrier means more DNA strands need to be synthesized with substantial investments.
Although DNA molecules are intrinsically biocompatible polymers and play very important roles in many biological and cellular processes, they may also elicit severe immune responses upon interactions with nearby cells and tissues which may further limit their clinical applications.
Over the past four decades, significant progress has been made in DNA nanotechnology, with promising results and proposals. However, no DNA-based technology or drug delivery system has yet received approval from the FDA, primarily due to the challenges outlined above. Additionally, navigating the regulatory challenges associated with novel DNA-based therapies is complex and time-consuming. Although DNA material is biodegradable, comprehensive assessments of their biosafety in humans are essential prior to real clinical applications. While the potential of DNA nanomaterials in drug delivery is rapidly advancing with ongoing research and clinical trials, most studies have been conducted on cells, tissues, or tumor-bearing mice. There remains a considerable journey ahead to translate these promising results into practical clinical applications. Overcoming these obstacles is crucial for the successful development and application of DNA nanostructure-based drug delivery systems in humans.
References
l. The Helicity of a DNA-2’-Fluoro DNA Hybrid Duplex Structure. Int. J. Nanotech. Nanomed. 2017; 2, 1-3.
2. Seeman, NC. DNA in a material world. Nature. 2003; 421, 297-302.
3. Seeman, NC. Nucleic acids junctions and lattices. J. Theo. Biol. 1982; 99, 237-247.
4. Weiss, PS. A Conversation with Prof. Ned Seeman: Founder of DNA Nanotechnology. ACS Nano. 2008; 2, 1089-1096.
5. Chen, J. and Seeman, NC. The synthesis from DNA of a molecule with the connectivity of a cube. Nature. 1991; 350, 631–633.
6. Mao, C. et al. A DNA nanomechanical device based on the B-Z transition. Nature. 1999; 397, 144–146.
7. Robinson, BH. and Seeman, NC. Simulation of double-stranded branch point migration. Biophysical J. 1987; 51, 611-626.
8. Madhanagopal, BR. et al. DNA Nanocarriers: Programmed to Deliver. Trends in Biochem. Sci. 2018; 43, 997–1013.
9. Xu, F. et al. Rationally Designed DNA Nanostructures for Drug Delivery Front. Chem, 2020; 8:751.
10. Chandrasekaran, AR. Nuclease resistance of DNA nanostructures. Nature Rev. Chem. 2021; 5, 225–239.
11. Rothemund, PWK. Folding DNA to Create Nanoscale Shapes and Patterns. Nature. 2006; 440, 297-302.
12. Zheng, J. et al. From molecular to macroscopic via the rational design of a self-assembled 3D DNA crystal. Nature. 2009; 461, 74–77.
13. Winfree, E. et al. Design and Self-Assembly of Two-Dimensional DNA Crystals. Nature. 1998; 394, 539-544.
14. Yurke, et al. A DNA-fuelled molecular machine made of DNA. Nature. 2000; 406, 605–608.
15. Chang, M. et al. Aptamer-conjugated DNA icosahedral nanoparticles as a carrier of doxorubicin for cancer therapy. ACS Nano. 2011; 5, 6156–6163.
16. Bhatia, D. et al. A synthetic icosahedral DNA-based host-cargo complex for functional in vivo imaging. Nat. Commun. 2011; 2, 339.
17. Liu, J. et al. A self-assembled platform based on branched DNA for sgRNA/Cas9/antisense delivery. J. Am. Chem. Soc. 2019; 141, 19032-19037.
18. Li, B. et al. Nano-drug co-delivery system of natural active ingredients and chemotherapy drugs for cancer treatment: A review. Drug Deliv. 2022; 29, 2130–2161.
19. Zeng, YC. et al. Fine tuning of CpG spatial distribution with DNA origami for improved cancer vaccination. Nat. Nanotechnol. 2024; 19, 1055–1065.
20. Kim, KR. et al. Drug delivery by a self-assembled DNA tetrahedron for overcoming drug resistance in breast cancer cells. Chem. Commun. 2013; 49, 2010–2012.
21. Li, J. et al. Self-assembled multivalent DNA nanostructures for noninvasive intracellular delivery of immunostimulatory CpG oligonucleotides. ACS Nano. 2011; 5, 8783–8789.
22. Xia, Z. et al. Tumor-penetrating peptide-modified DNA tetrahedron for targeting drug delivery. Biochem. 2016; 55, 1326– 1331.
23. Lee, H. et al. Molecularly self-assembled nucleic acid nanoparticles for targeted in vivo siRNA delivery. Nat. Nanotechnol. 2012; 7, 389–393.
24. Ora, A. et al. Cellular delivery of enzyme-loaded DNA origami. Chem. Commun. 2016; 52, 14161–14164.
25. Wu, C. et al. Building a multifunctional aptamer-based DNA nanoassembly for targeted cancer therapy. J. Am. Chem. Soc. 2013; 135, 18644–18650.
26. Qu, Y. et al. Self-assembled DNA dendrimer nanoparticle for efficient delivery of immunostimulatory CpG motifs. ACS Appl. Mater. Interfaces 2017; 9, 20324–20329.
27. Kim, E. et al. One-pot synthesis of multiple protein encapsulated DNA flowers and their application in intracellular protein delivery. Adv. Mater. 2017; 29, 1701086.
28. Jiang, Q. et al. Self-assembled DNA origami-gold nanorod complex for cancer theranostics. Small 2015; 11, 5134–5141.
29. Chen, X. et al. Triplex DNA Nanoswitch for pH-Sensitive Release of Multiple Cancer Drugs. ACS Nano 2019; 13, 7333−7344.
30. Bujold, KE. et al. Optimized DNA nano-suitcases for encapsulation and conditional release of siRNA. J. Am. Chem. Soc. 2016:138, 14030–14038.
31. Zeng, Y. et al. Time-lapse live cell imaging to monitor doxorubicin release from DNA origami nanostructures. J. Mater Chem. 2018; B 6, 1605–1612.
32. Sun, W. et al. Self-assembled DNA nanoclews for the efficient delivery of CRISPR-Cas9 for genome editing. Angew. Chem. Int. Ed. Eng. 2015; 54, 12029–12033.
33. Kumar, M. et al. DNA-Based Nanostructured Platforms as Drug Delivery Systems. Chem Bio Eng. 2024; 1, 179−198.
34. Kosara, S. et al. Structural DNA nanotechnology at the nexus of next-generation bio-applications: challenges and perspectives. Nanoscale Adv., 2024; 6, 386
35. Zhang, Y. et al. Advanced applications of DNA nanostructures dominated by DNA origami in antitumor drug delivery. Front. Mol. Biosci. 2023; 10:1239952
36. Allentoft, M. E. et al. The half-life of DNA in bone: measuring decay kinetics in 158 dated fossils. Proc. R. Soc. B Biol. Sci. 2012; 279, 4724–4733.
37. Tian, T. et al. Prospects and challenges of dynamic DNA nanostructures in biomedical applications. Bone Research. 2022; 10, 40.