Figure 1: DNA nanodevices identifying and analyzing cancer cells

Applications of DNA Nanotechnology in Cancer Diagnosis

Introduction DNA, which is responsible for the storage and transfer of genetic information, can transition from a single-stranded to a double-stranded structure according to the Watson-Crick base pairing principle, making it a promising candidate for creating complex nanoscale architectures (1). It has high biocompatibility due to its low immunogenicity compared to other nanomaterials, can spontaneously assemble into various nanostructures through well-designed protocols due to its high programmability, can form stable structures in vivo by resisting nucleases, and can easily penetrate tissues when arranged in tetrahedral structures (2). Currently, cancer diagnosis mostly relies on radiological imaging methods, tissue biopsies, and detection of certain tumor markers in patients’ bodily secretions such as blood, urine, and saliva. In recent years, efforts have been made to develop liquid biopsy techniques, biosensors, and nanoprobes that can be used in cancer detection by taking advantage of DNA’s molecular recognition capabilities, programmability, and self-assembly. Furthermore, various DNA nanotechnology methods, such as DNA origami, self-assembling DNA nanostructures, and hybrid nanocomplexes, are used for the recognition and amplification of biomarkers in cancer detection. This article will discuss nanotechnology methods used in cancer diagnosis.

DNA Nanostructures DNA can be assembled into various nanostructures such as origami, tetrahedrons, nanoflowers, hydrogels, and nanospheres. In DNA origami, a long single-stranded DNA molecule is folded into a specific geometric shape using shorter complementary DNA strands. Each strand of the DNA helix within DNA origami can be engineered to enable highly specific modifications. Furthermore, due to DNA s self-assembly property, billions of DNA origami structures can be folded simultaneously within a very small volume. Its ability to selectively bind to molecules enables its use as a nanoscale carrier for targeted delivery. These properties make DNA origami promising in biomedicine and cancer treatment. DNA tetrahedrons are tetrahedral structures generated through the self-assembly of DNA sequences (3). Thanks to their structural properties, they can be used as versatile nanocarriers and may be particularly effective in tumor treatment (4). DNA nanoflowers are formed by the disordered self-assembly of DNA. These nanostructures can find applications in biosensoring, biological imaging, and therapeutic interventions. They may reveal significant potential for tumor diagnosis and treatment (5). DNA hydrogels exhibit well-defined architectures, tunable mechanical properties, and multiple interaction sites, which contribute to their high biocompatibility and structural stability. They are considered particularly promising nanostructures for disease diagnosis and drug delivery (6,7). DNA nanospheres are also nanostructures that can be widely used for biological imaging and tumor treatment (8). Nucleic acid aptamers are nanostructures composed of DNA or RNA oligonucleotides that adopt various 3D configurations and recognize specific targets with high binding affinity (9). They have advantages such as programmability, low immunogenicity, ease of synthesis, and modification. They have a wide range of target recognition capabilities, including small molecules, proteins, different metal ions, and various tumor markers (10–13).

DNA Nanotechnology in Cancer Diagnosis In recent years, DNA nanotechnology has been used in cancer diagnosis, particularly for the recognition and amplification of biomarkers. MicroRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) are being tested as biomarkers in tumor diagnosis (14,15). A method using target-induced chain amplification has enabled reliable detection of selected miRNAs even at single-molecule levels inside tumor cells (15). A method utilizing a DNA tetrahedron has been developed for more sensitive detection of miRNAs. In this method, a DNA tetrahedron activated via catalytic hairpin assembly functions as an electrochemical biosensor, and upon encountering miRNA-141, initiates an enzyme-free cycle, leading to an increase in the electrochemical signal in the environment. This increases sensitivity and allows for faster recognition of tumor markers (16). In another method, intracellular miRNA sequences were attempted to be detected by designing a tetrahedral DNA framework. This tetrahedral framework can facilitate the identification of additional target genes in cells by allowing modification of the DNA sequence (17). In addition, exosomes are quite promising as non-invasive cancer biomarkers. The most important disadvantages are the difficulties in detecting them with high sensitivity and accuracy. Unlike traditional tissue biopsies, liquid biopsy allows the detection of biological markers such as circulating tumor DNA (ctDNA), tumor cells (CTCs), and extracellular vesicles (18, 19). ctDNA refers to tumor DNA released into the bloodstream by necrotic or apoptotic tumor cells. It provides information about the genetic structure of the tumor. It can be very useful in the early diagnosis of cancer and monitoring of treatment response (20, 21). However, their presence in circulation at very low concentrations makes their detection difficult using classical methods. To overcome this difficulty, DNA nanotechnology-based methods are being developed. In lung cancer patients, ctDNAs were identified and signal amplifications were increased using DNA-rN1-DNA-mediated surface-enhanced Raman scattering, after which ctDNA testing was performed in serum samples taken from these patients (22). In breast cancer cases, tetrahedral DNA nanostructures were integrated with carbon nanotubes to enable the detection of ctDNA related to the AKT2 gene (23). Additionally, the detection of carcinoembryonic antigen was achieved using a DNA nanoflower system (24). In recent years, DNA-enhanced nanoprobes including gold nanoparticles and silver nanoclusters have been useful in detecting mutated ctDNA (25). Non-invasive treatment monitoring has been achieved in patients undergoing immunotherapy by employing gold nanorods modified with complementary DNA probes for ctDNA analysis (26). Nanosensors constructed using DNA origami are also used to facilitate ctDNA measurement. A DNA origami method functionalized with aptamers has been tried for the detection of glioblastoma-associated ctDNA mutations, and more sensitive measurement of ctDNA has been achieved compared to PCR-based methods (27). Quantum dot-based DNA biosensors have also been used to detect ctDNA mutations in cancer screenings (28). Another liquid biopsy marker that can be used in cancer diagnosis is CTCs. In recent years, research has been conducted on DNA nanostructures functionalized with aptamer-based recognition probes for the detection of CTCs (29). Thanks to DNA-based biosensors, very low concentrations of cancer biomarkers can be detected, which allows for early diagnosis of cancer before clinical symptoms appear. This is useful in assessing the risk of metastasis progression. Matrix metalloproteinase 2/9 (MMP2/9) and ATP are metastasis-related targets in the extracellular tumor microenvironment. Scientists have engineered a DNA nanodevice containing an ATP-sensitive aptamer sensor and a MMP2/9 degradable peptide-nucleic acid copolymer to detect these markers. This allows for the simultaneous detection of several biomarkers indicating tumor metastasis (30). One of the biomarkers of malignant tumors is proteases expressed from cancer cells. By employing a strategy in which a nucleic acid peptide nucleic acid copolymer stabilizes the aptamer structure, protease selectively activates the aptamer probe, generating tumor-specific fluorescent signals and enabling sensitive in vivo molecular detection (31). Researchers have developed a light-sensitive DNA origami structure containing a protein coating consisting of both a targeting component and a camouflage component for the detection of the HER2 biomarker used in breast cancer diagnosis. In this method, after light irradiation, the coating breaks down, the camouflage protein is released into the environment, and the targeting part specifically recognizes HER2 (32). DNA-based nanoprobes are among the developments that could revolutionize tumor imaging. These nanoprobes are capable of selectively recognizing tumor-associated markers through DNA aptamers, hybridization chain reactions, or DNA-templated nanoparticles, thereby improving image resolution, specificity, and overall image quality. They can enable tumor imaging by acting as highly selective and targeted contrast agents across different imaging modalities such as fluorescence imaging, magnetic resonance imaging, and computed tomography (33,34). Real-time and high-resolution monitoring of cancerous tissues is possible with fluorescent DNA nanoprobes (35). By increasing the contrast of magnetic resonance imaging with DNA-functionalized superparamagnetic iron oxide nanoparticles, the detection of deeply located tumors can be made easier (36). In addition, in optical and photoacoustic techniques, excellent plasmonic properties can be achieved and imaging sensitivity can be increased by using gold nanoparticles conjugated with DNA strands. Conclusion DNA nanotechnology can enable the early and highly accurate detection of cancer, allowing for timely and effective intervention. Thanks to advantages such as excellent biocompatibility, programmability, and ease of modification and functionalization, DNA nanostructures can lead to new methods in biosensors and biological imaging that will facilitate cancer diagnosis and treatment. However, for this technology to fully take its place in clinical applications, various challenges such as large-scale production, stability in physiological environments, and long-term biocompatibility need to be re-evaluated. Despite these challenges, advancements in DNA nanotechnology indicate that DNA nanomaterials will play a greater role in clinical applications in the future.

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