Recent advances in DNA nanomaterials for cancer diagnosis and treatment

2.1 Development of DNA nanomaterials

The development of DNA nanotechnology began with a concept proposed by Seeman in 1980, who realized that the branching connection in DNA can be used to form three-dimensional (3D) crystal materials through their sticky ends. Seeman envisaged assembling branching connections into 3D crystals and using them as 3D scaffolds for proteins and other biological components, thereby solving the macromolecular crystallization problem [19]. However, DNA branching junctions (such as four-arm Holliday junctions) are limited by their geometric flexibility and instability. In 1983, Seeman and Gang stabilized a Holliday junction by manufacturing arms with unique sequences, constructed a 3D DNA cube in the following decade, and reported double-cross (DX) molecules composed of two exchange-connected DNA double-helix strands; these DX molecules are geometrically rigid and stable. Subsequently, DX molecules were also designed to assemble into two-dimensional (2D) DNA crystals, and many 2D and 3D DNA structures constructed using DNA tiles were reported between 1980 and 1998; these structures can also be used as dynamic molecular-machine components [19].

A three-dimensionally structured DNA was first proposed by Chen and Seeman in 1991 [20], with the DNA tetrahedron gradually becoming a typical 3D structure following development and simplification; it still plays important roles in detection and analysis applications, and as drug carriers. The introduction of modular construction methods led to the development of more complex polyhedral DNA nanocage structures. DNA dendrimers, hydrogels, and other 3D functional nanostructures or construction methods have also sequentially appeared, thereby enriching the advanced DNA structure family. DNA origami technology has expanded the library of 3D DNA structures, and the construction of various 3D structures, including DNA boxes, vases, gears, rabbits, and robots, has become increasingly easy [19].

Based on previous studies, in 2006 Rothemund creatively introduced revolutionary DNA origami technology that changed the face of DNA nanotechnology [21]. DNA origami is a class of technologies for building DNA nanostructures by folding a long single-stranded DNA (ssDNA; scaffold) into desired shapes via base pairing. The basic idea is to scaffold a single strand of DNA that is a long circular strand (called the scaffold strand, typically the circular DNA of phage M13mp18, with a length of 7,429 bp). Specialized design software is used to produce large quantities of short strands of DNA from the base sequences of the scaffolding strands as staple strands, which are complementary to the specific base sequence of the scaffold strands. More than 200 staple chains with different sequences are paired with scaffold strands in different positions via the simple operation of heating and annealing, and the distant bases are pulled closer, so that the long chain is folded with preset shape. The structure of the DNA origami can be changed by altering the base sequence of the staple strands.

2.2 Characteristics of DNA nanomaterials

DNA nanostructures have been shown to exhibit a lack of toxic side effects, low immunogenicities, and good cell/serum stabilities; they can also be transported across membranes, which are basic drug-carrier conditions. DNA hydrogels have been used to deliver small drug molecules and proteins to cells [22], DNA tetrahedrons can be designed as carriers for immune-enhancing drug by enhancing stability and targeting [23], and the DNA double helix structure is the insertion site of some chemotherapeutic small drug molecules (such as DOX), which facilitates delivering a large number of chemotherapeutic drugs into tumor cells [24]. Short DNA strands can also be extended on a DNA vector to complement and pair with small interfering RNA (siRNA), and to carry them into cells in order to change target-protein expression [25,26].

Moreover, the nucleic acid material is highly operable: precise DNA nanostructures can be designed and implemented at nanoscale [27,28]. In addition, specific insertion and deletion of bases can control the stress and flexibility of DNA nanostructures to build complex forms with curves [29]. The synthesis and modification of DNA have been commercialized, the desired designed sequence can easily be obtained in a timely manner, and the tool enzymes that act on DNA are abundant and diverse. Methods that use branched DNA and nucleic acid aptamers have also delivered good molecular recognition and disease diagnosis results [30].

2.3 Strategies of preparation and regulation for DNA nanomaterials

DNA is an exceptional nanomaterial owing to the advantages presented above. DNA nanomaterials with increasingly complex structures and superior functions have been continuously designed through the development and optimization of DNA tile assemblies, DNA origami, branched-DNA assemblies, rolling ring amplification, isothermal amplification, and other technologies [7,31–46] (Figure 1).

Figure 1

Development history of nucleic acid nanotechnology. (a) DNA double helix – 1953. Branched DNA (b–g): (b) branched DNA – 1983, (c) metal nanobranched DNA conjugates – 2005, (d) branched DNA self-assembled to form i-motif – 2009, (e) branched DNA linked by linker DNA – 2010, (f) branched DNA PCR primer – 2013, (g) branched for detection of liver cancer – 2018. DNA origami (h–n): (h) DNA origami nanotechnology – 2006, (i) 3D DNA origami – 2009, (j) 2D DNA origami arrays – 2010, (k) complex curvatures 3D DNA origami – 2011, (l) DNA origami guides the growth of AuNPs – 2014, (m) DNA origami conjugated polymer assembly – 2016, (n) DNA nanorobot for cancer treatment – 2018. DNA hydrogel (o–u): (o) oligonucleotide hydrogel – 1996, (p) enzyme-catalyzed assembly of DNA hydrogel – 2006, (q) temperature-controlled hydrogel – 2008, (r) self-assembled DNA hydrogels – 2010, (s) photosensitive DNA hydrogel for cancer treatment – 2011, (t) shape-memory DNA hydrogels – 2014, (u) cell-laden DNA hydrogel assemble brick tissue-like structures – 2017. Reprinted with permission from refs [7,31–46]. Copyright Wiley-VCH and American Chemical Society.

One-, two-, and three-dimensional materials with various structures can be prepared using current mature synthesis methods like branched DNA [44] (Figure 2). However, cancer diagnosis and treatment always require structural reconstruction to meet application requirements for specific scenarios, with DNA hydrogel formation and disintegration before and after circulating tumor cell (CTC) capture, and the release of drug carriers after carrying drug molecules to target positions, as examples. Therefore, some regulatory strategies are needed to control nucleic acid nanomaterial reconstruction.

Figure 2

Overall types of branched DNA building blocks and construction methods of branched DNA-based materials. Reprinted with permission from ref. [30]. Copyright 2020 American Chemical Society.

Chandrasekaran et al. [47,48] summarized the following seven common regulation strategies (Figure 3). (a) Regulating the toehold-mediated chain displacement reaction: the toehold region reserved in a DNA nanomaterial can be regulated by chain displacement reactions. (b) DNA short chain regulation: a short ssDNA can change the structure of the DNA nanostructure when a hairpin is included. (c) pH regulation:some special short DNA chains respond to pH changes (for example, a sequence rich in cytosine deoxyribonucleotide can form a tetramer i-motif under acidic conditions, which can be designed into the DNA structure to regulate structural changes. (d) Regulating the nucleic acid aptamer: a nucleic acid aptamer can be designed into the DNA nanomaterial through complementary base pairing in the absence of an antigen. When bound by the target antigen, the conformation of the nucleic acid aptamer changes significantly and breaks away from the main DNA structure, resulting in nanomaterial reconstruction. (e) Photo-control involving connecting a crosslinking agent that is easily photodegraded to the DNA nanomaterial and changes its structure when exposed to light. (f) Regulating the temperature: some hairpin structures are sensitive to temperature, and reversibly adjusting the temperature can regulate the reconstruction of the DNA structure. (g) Regulating the telomerase primer: tumor cells are rich in telomerase, and telomerase primers become extended when the nanomaterial enters a tumor cell, resulting in structural changes when the DNA nanomaterial is designed to include telomerase primer sites and telomerase repeats.

Figure 3

Regulation strategies for reconfiguring a DNA nanomaterial. Reprinted with permission from ref. [47]. Copyright 2019 American Chemical Society. (a) Toehold-based strand displacement. (b) Short strand DNA regulation. (c) pH regulation. (d) Aptamer-based regulation. (e) Light regulation. (f) Temperature regulation. (g) Telomerase activity regulation.

Various regulatory elements can be introduced into the system using the abovementioned strategies, so as to optimize the performance of the nucleic acid nanomaterial, improve its environmental response function and controllability, and provide a foundation for building intelligent nucleic acid nanomaterials.

The rapid development of research into nucleic acid nanomaterials has led to increasingly more nucleic acid nanomaterials used in biosensor, molecular-detection, cell-imaging, drug-delivery, and nano-photonics research, among others.

3.1 Branched DNA nanomaterials in cancer diagnosis and treatment applications

As a general molecular component used to build functional materials, the topological structures of artificial DNA polymer chains include linear, cyclic, and branched forms [49], while linear and cyclic DNA are the most common naturally occurring forms. Linear DNA is the most easily designed and synthesized; consequently, it is often used as the basic unit for constructing various nanomaterials [50]. Cyclic DNA can also be used as a scaffold for constructing some DNA nanostructures, such as rigid triangles [51], rotane-like DNA structures [52], and DNA nanotubes [52]. However, the use of circular DNA as a component is associated with certain limitations. On the one hand, double-stranded DNA (dsDNA) is a relatively rigid polymer from which small circular dsDNA is difficult to form using only base stacking forces [51,52]. On the other hand, some organic molecules or enzymes need to be introduced to form small circular DNA [53,54], which increases operational complexity. Consequently, branched structures need to be introduced in order to form multidimensional DNA nanostructures. Branched DNA has a more complex topological structure and functional modules that overcome some of the limitations of linear DNA and circular DNA. Branched DNA can (a) act as initiator cores or building-block units for growth when constructing hyperbranched DNA nanostructures in a controlled manner, (b) its sequence can be reasonably designed according to needs, and (c) custom sticky ends on branched DNA provide multiple modification sites that can be linked to specific functional parts [55,56]. Reasonably designed branched DNA monomers are capable of generating high-order 2D or 3D DNA nanostructures [30], including DNA dendrimers [57], nanonets [58], polyhedrons [59], crystals [60], and hydrogels [61], through sticky-end self-assembly (Table 1).

Branched DNA not only has the ability to identify a variety of target molecules accurately and rapidly, its unique multi-arm structure can also be used as a multifunctional signal amplifier to deliver rapid, sensitive, personalized, and multifaceted diagnosis [70–76]. Many molecular-detection methods based on branched DNA have been developed by incorporating branched DNA into various amplification strategies. For instance, a study demonstrated the use of branched DNA nanomaterials as contrast agents for magnetic resonance imaging (MRI) of prostate cancer, leading to improved tumor detection [77]. Li et al. [78] described herein a single-step and highly sensitive method, termed aptamer-triggered cascade primer exchange reaction (PER)-generated branched DNA nanostructures, for the quantification and imaging of exosomal PD-L1. The presence of exosomal PD-L1 converted the conformation of the recognition probe, accompanied by the exposure of primer 1. Then, primer 1 actuated the cascade PER, which generated branched DNA nanostructures containing numerous guanine quadruplex (G-quadruplex) for binding to thioflavin T dye, leading to an amplified fluorescence signal. Profiting from directly growing branched DNA nanostructures on the surface of exosomes, the size of exosomes was enlarged and the movement of exosomes was limited, achieving the imaging of exosomal PD-L1 by conventional optical microscopy in a wash- and label-free fashion. Analyzing exosomal PD-L1 from serum samples of 15 cancer patients and 15 healthy volunteers demonstrated that this simple strategy could distinguish non-small cell lung cancer patients from healthy donors with high clinical accuracy. Liu et al. [79] presented a spatial confinement Förster resonance energy transfer (SC-FRET) probes with stable structure and strong signal output. The proposed SC-FRET system based on the multivalent assembly of branched DNA displayed strong FRET response with high quantum yield and showed a high sensitivity with limit of detection of 0.01394 pM. Importantly, it also showed a high-contrast and stable FRET response in HeLa cells. Its superior biological stability is attributed to the large steric hindrance of the compact and rigid frame of the SC-FRET probe, which helps prevent intracellular degradation and provides a powerful tool for biomedical research. Cheng et al. [8] exploited the release of electroactive hybridization chain reaction (HCR) and mesoporous silica nanoparticle substances activated by coupling targets to detect tumor-related messenger RNA (mRNA) in an ultrasensitive manner (Figure 4a). The platform hybridizes with the target mRNA recognition probe terminated by a 5′-phosphate when introduced into the target, thereby opening the DNA gate that releases electrically active myoglobin. Using thymidine kinase 1 (TK1) mRNA as a model target, this strategy quantitatively detected TK1 mRNA with a 0.1 fM to 1 pM detection range and a 2.0 aM detection limit. Compared with reported electrochemical RNA sensing technology, the developed electrochemical sensing platform has the advantages of high sensitivity, lack of label, and low background signals.

Figure 4

(a) Schematic diagram showing an electrochemical sensing platform for low background cascade signal amplification. (b) SERS biosensor based on branching DNA. (c) Branch DNA cell level detection method. (d) Branched antisense and siRNA for combined gene silencing. Reprinted with permission from refs [7,8,80,81].Copyright (2018) American Chemical Society, (2021) Frontiers in Immunology and (2020) Wiley-VCH.

The abovementioned platform has reportedly been used to detect liver cancer. Cheng et al. [7] designed an ultrasensitive surface-enhanced Raman scattering (SERS) biosensor based on the branched-DNA-assisted “sandwich” strategy. The branched DNA consisted of a ssDNA sequence as the active adhesive end, and three branches (Y-DNA) with a dsDNA core as the scaffold (Figure 4b). This system is capable of detecting miRNAs and proteins in complex physiological environments. Aided by micro-contact printing technology, this system can easily simultaneously detect miRNAs and the established alpha-fetoprotein (AFP) liver-cancer biomarker at the molecular level.

Garcia-Perez et al. [80] also invented a cell-level detection method that combines branched DNA technology with flow cytometry; the method is capable of directly measuring mRNA to evaluate single-cell gene transduction and expression levels, and is more sensitive than quantitative PCR for detecting small numbers of positive cells (Figure 4c). Wu et al. [82] assembled DNA-templated silver nanoclusters (DNA AgNCs) through a multi-branched linear (MBL) DNA structure formed by a triggered HCR. DNA AgNCs assembled by the MBL structure tethered to aptamers that recognize cancer cells exhibited detection sensitivity about 20 times greater than that observed for DNA AgNCs tethered by single aptamers. Aided by an “AND” logic platform, DNA AgNCs assembled by doubly attached MBL exhibited logic performance for analyzing double-cell surface receptors; consequently, they have the ability to identify cancer cells highly sensitively and with high resolution. Branched DNA nanomaterials have been used as theranostic agents, combining diagnostic and therapeutic functions. Liu et al. reported the development of branched DNA nanomaterials functionalized with aptamers for targeted delivery of photodynamic therapy agents and imaging probes to cancer cells, leading to enhanced therapeutic efficacy and imaging sensitivity [82].

The excellent properties of DNA (biocompatibility, low immunogenicity, and stability) engender branched DNA with broad applications prospects for the development of new cancer-treatment materials. Combined with the high loading capacity of branched DNA, branched-DNA/aptamer-based nanomaterials that carry anticancer drugs are cytotoxic to the target cancer cells and exhibit few side effects to non-target cells [83,84]. Developing nanocarrier systems with sufficient drug-loading capacities and efficient drug-release behavior in cells is a powerful strategy for maximizing the therapeutic effect and reducing the side effects associated with drug administration. Bi et al. [85] introduced novel nanospheres based on branched DNA as drug delivery carriers. The nanospheres have the advantages of uniform size, good monodispersity, compactness, and multi-functional cantilevering. Branched DNA nanomaterials can be used to load drugs and also participate in drug release and regulation. Nishikawa et al. [86,87] first showed that, compared to traditional dsDNA and ssDNA, Y-shaped DNA that contains the cytosine–guanine dinucleotide (CpG) motif significantly increases cytokine secretion from RAW264.7 macrophages, while branched DNA with more arms biodegrade better and are more immunostimulatory [88]. Ma et al. reported functional branched DNA nanomaterials with targeting ligands to deliver DOX to HER2-positive breast cancer cells, resulting in increased drug uptake and reduced cytotoxicity to normal cells [89].

Some nucleic acid drug molecules produce unexpected effects when combined with branched DNA. In 2019, Liu et al. [81] reported the use of the cyclic β-cyclodextrin supramolecule (as the core) to covalently crosslink nucleic acids using copper-free click chemistry and prepare branched DNAs with a seven-arm structure (Figure 4d). First, covalently binding the branched antisense effectively captures 3′-siRNA to form a NP with controllable size. Second, biocompatible binuclear acidic nanostructures can be functionalized by exploiting host–guest interactions with targeted groups and endoplasmic escape components. Finally, multifunctional nucleic acid nanocarriers are digested by endogenous ribonuclease (RNase) H to effectively release nucleic acid products (branched antisense and siRNA). This new strategy skillfully combines DNA and a peptide through nucleic acid self-assembly and host–guest interactions, to realize a multifunctional nucleic acid carrier and provide a new nucleic acid transmission concept. Branched DNA nanomaterials have also been explored as gene editing tools for cancer treatment. Liu et al. used branched DNA as carriers for CRISPR/Cas9 gene editing complexes to selectively target cancer cells, resulting in effective inhibition of tumor growth [90].

The flexible ends of branched DNA can elongate its structure to form DNA networks or connect multiple functional elements, thereby creating conditions for various biomaterial and biomedical applications [30]. Unlike conventional chemical dendrimers with isotropic structures [84], branched DNA can be designed to be isotropic or anisotropic, and symmetric or asymmetric; hence, it is flexible, universal, and an ideal building block [91].

3.2 DNA hydrogels for cancer diagnosis and treatment

Nagahara and Matsuda [92] first introduced the concept of composite DNA hydrogels in 1996, which were initially constructed by modifying DNA strands on a polyacrylamide skeleton. These DNA polymer composite hydrogels are highly biocompatible and maintain the specific recognition ability of DNA. Subsequently, DNA polyacrylamide copolymers were used to construct various functional hydrogels using similar strategies. However, the process used to modify DNA polymer hybrid chains is cumbersome, which limits their development to some extent. In 2006, Um et al. [93] connected the sticky ends of branched DNA assemblies using T4 ligase and constructed a hydrogel composed entirely of DNA. This hydrogel shows good shape-memory performance and can be loaded with drugs, proteins, and even living cells; these substances are released gradually as the DNA degrades. The use of DNA hydrogels in biomedical applications has flourished since this initial work.

A DNA hydrogel network provides an elastic, semi-humid, and 3D environment. These characteristics are suitable for cell adhesion and have widespread use in 3D cell-culturing, tissue-engineering, CTC-capturing, and analysis applications. Song et al. [94] reported the preparation of porous DNA hydrogels using an aptamer trigger clamped HCR (atcHCR) method (Figure 5a) for the in situ recognition and capture of CTCs. The entire process is initiated by the EpCAM protein on the surface of the CTC, which prepares the nucleic acid aptamer initiator Bi block, triggers the atcHCR reaction, and assembles hydrogels around the CTC to fix it. A DNA hydrogel contains an adenosine 5′-triphosphate (ATP) responsive region. The addition of ATP destroys the hydrogel structure and releases the CTC. This method may provide a new liquid-biopsy-based path for the early screening of cancer. Jin et al. [95] report a dual-targeted aptamer-decorated DNA hydrogel system (DTA-H) to achieve efficient, stable, and targeted delivery of drugs. First, DNA hydrogel was formed by the RCA. By reasonable design, double target and multivalent aptamers were decorated on DNA hydrogel to load DOX. The results confirmed that DTA-H can deliver chemotherapy drugs and aptamer nucleic acids drugs to target cells, inducing degradation of HER2 protein while chemotherapy is synergistic to inhibit HER2-positive breast cancer growth. The proposed drug delivery system has significant potential to achieve efficient, stable, and targeted delivery of drugs and cancer therapy.

Figure 5

(a) Application of DNA porous hydrogel in cancer diagnosis. (b) DNA hydrogel electrochemical biosensor. (c) DNA hydrogel SERS biosensor platform. (d) Injectable DNA hydrogel for chemotherapy. Reprinted with permission from refs [94,97–99]. Copyright American Chemical Society.

DNA hydrogels have attracted increasing levels of attention in the sensing and medical treatment fields as they are naturally biocompatible and mechanically stable. While electrochemical sensing technology is both sensitive and quantitative, electrochemical DNA hydrogel biosensors have rarely been reported. In this space, Liu et al. [96] reported an electrochemical biosensor based on a hybrid DNA hydrogel immobilized on an ITO/PET electrode (Figure 5b) for detecting miR-21, a lung cancer specific microRNA. Mao et al. [97] successfully prepared 3D electron transporters on traditional electrodes using electrode-supported DNA hydrogels as network scaffolds. Compared with the traditional nonfunctional electrode, the 3D electron transporter delivered a better electron-transfer effect and a more-intense output signal.

Despite the simple and direct visual sensing of various cues, sensitivity and quantification obstacles have limited the usage of DNA hydrogels. Therefore, designing intelligent gels that quickly respond to macromolecular input is a major challenge. Khajouei et al. [100] found by taking advantage of signal amplification mechanisms such as the amylase/iodide system and cascade reactions, the sensitivity can be significantly enhanced. This can strengthen the application of DNA hydrogel in biosensor. To improve the accuracy of DNA hydrogels for detecting targets, Yao et al. [101] reported the design of pH-responsive DNA hydrogels with ratiometric fluorescence. The DNA hydrogels were prepared from the pH-sensitive ZnO-NH₂ and CO-Y-DNA probe assembled by the three complementary strands. With the use of miRNA-21 as the model analyte, the DNA hydrogels were applied to fluorescence ratio detection. Under acidic conditions, the ZnO-NH₂ was decomposed, thereby releasing the CO-Y-DNA probe. Target miRNA-21 hybridized to the CO-Y-DNA probe, causing the change of fluorescence ratio between TAMRA and Cy5 that both modified in the CO-Y-DNA probe. The developed DNA hydrogels exhibited high accuracy and sensitivity with a low detection limit to 83 pM.

Wang et al. [98] designed and constructed a new SERS biosensor platform (Figure 5c) in combination with an aptamer-responsive DNA hydrogel for the sensitive detection of AFP. The encapsulating ability of the DNA hydrogel was first exploited to embed IgG; the AFP aptamer specifically recognizes AFP, forms a target aptamer complex that triggers the dissociation of the network structure of the hydrogel and releases the embedded IgG. The released IgG then binds to the antibody attached to the nanoprobe and the capture probe. SERS probes and biofunctional magnetic beads capture the released IgG, which leads to a less-intense Raman-tag signal from the supernatant following magnetic separation. Since hydrogel dissociation is directly controlled by AFP, this method can be used to quantitatively detect AFP. The method exhibits a low detection limit of 50 pg/mL, is inexpensive, rapid, and sensitively detects AFP.

In addition to detecting liver- and lung-cancer markers, DNA hydrogel materials can also be used to diagnose gender-specific cancers, such as breast, cervical, and prostate. For example, Chen et al. [9] constructed a real-time solid-phase reversible immobilization biosensor for sensitively detecting prostate cancer exosomes. The hydrogel not only avoids the agglomeration of embedded AuNPs, but also effectively amplifies SPR signals by taking advantage of its porous structure and large specific surface area. Sun et al. [10] constructed a DNA hydrogel network (2h-DNH) using a cross-linked hybridization chain reaction (c-HCR) mediated by palindrome ends. The c-HCR strategy can be used to screen intracellular miRNAs, with an almost two-to-three order-of-magnitude improvement in detection sensitivity. Diseased cells (HeLa) were distinguished from healthy cells (HEK-293), and MCF-7 cells with different miR-21 levels were accurately identified.

Photothermal immunotherapy has become a very effective cancer-treatment method; however, this strategy is impacted by the poor biodegradability of photosensitive materials. In one study, Wu et al. [102] aimed to develop biodegradable materials for photothermal immunotherapy; to this end, a DNA CpG hydrogel (DH, generated by RCA) was prepared, loaded with bis(3′–5′)-cyclic dimeric guanosine monophosphate (G/DH), and coated with melanin (Mel/G/DH) during formulation. Mel/G/DH exhibited a higher temperature when illuminated with near infrared (NIR) light. Mel/G/DH + NIR (808 nm) irradiation exposed CT26 cancer cells to calcium in vitro and significantly promoted dendritic cell (DC) maturation. Local administration of Mel/G/DH + NIR triggered primary-tumor death through photothermal killing in vivo, and DC maturation in lymph nodes. Treatment of primary tumors with Mel/G/DH + NIR was found to reconstruct the immune microenvironment of distant tumors, increase the levels of cytotoxic T cells, and decrease the number of regulatory T (Treg) cells. In summary, this study revealed that Mel/G/DH is a potential platform for regulating the tumor immune microenvironment and can be used to prevent the recurrence of distant tumors in the future.

Zhang et al. [99] designed an injectable DNA hydrogel assembled using DNA chains grafted with chemotherapeutic drugs for local chemotherapy applications (Figure 5d). A large amount of CPT was first grafted onto the backbone of a DNA thiophosphate that can be assembled into two Y-shaped building blocks, and then layered and interconnected to form a drug-containing hydrogel. The hydrogel gradually decomposes into nanoparticles through enzymatic degradation; these particles efficiently penetrate into residual tumor tissue and are effectively absorbed by cells. The drug-containing DNA hydrogel exhibited sustained and responsive drug-release behavior and significantly inhibited tumor-cell regeneration and prevented cancer recurrence.

A hydrogel is a type of 3D networked soft material with a high water content (between that of a fluid and a solid) that is highly biocompatible, very structurally flexible, and multifunctional. Smart hydrogels are capable of sensing small physical and chemical changes (i.e., pH, temperature, light, heat, pressure, etc.) and respond rapidly. They are widely used in biochemical analysis, nanodrug delivery and controlled release, 3D cell culturing, and other applications [103].

3.3 DNA origami for cancer diagnosis and treatment

Since DNA origami was invention [21], its rapid development has the potential to greatly impact biology, chemistry, medicine, engineering, computational science, and other fields, with biomedical research aimed at intelligent applications being pursued. Due to precision and customizability, DNA origami structures can detect and diagnose various disease-related indicators, including pathogenic microorganisms, tumor markers, and gene mutation sites, among others. DNA origami carriers loaded with specific drugs can also be constructed to quantitatively release drugs at tumor-tissue target sites, which solves a current cancer (or other major disease, such as cardiovascular and cerebrovascular) treatment dilemma. The DNA origami structure can also be customized to form nanorobots capable of accurately and controllably completing various tasks according to the given instructions. In particular, medical nanorobots are important because they can perform tasks that cannot be completed using other medical bionics technologies [42,104,105]. DNA origami has nanoscale precision-addressing capabilities, which provide a structural basis for the design of spatial arrays with precise positions [106]. As an example, the DNA tile origami structure (DNA tile) is capable of providing hundreds of addressable sites that facilitate the design of nanoarray structures with different modes [107–109].

The rapid development of DNA synthesis and modification technologies has enabled the modification of many common functional groups, such as amino, carboxyl, hydroxyl, sulfhydryl, and fluorescent groups, at both ends of the DNA chain or at intermediate bases during DNA-chain synthesis. Therefore, indicating where the DNA strand should be modified before delivering it to the company for staple-chain synthesis that positions functional groups on the DNA-origami structure is only necessary [110]. The vigorous development of DNA-origami technology has also provided a new concept for constructing highly effective targeted drug carriers. Compared with the oligomeric short DNA single-strand or double-strand structure, the DNA-origami structure has a higher cell uptake efficiency, which provides a foundation for its use in nanodrug-loading applications. DNA-origami structures commonly used as nanodrug carriers include DNA tiles, triangles, cylinders, and hexagonal cylinders [111]. If properly designed, DNA origami technology can also have a great impact on the immune ability of cells. Kang et al. [112] found that the nanoparticle platform formed by combining DNA origami technology with nitrate (nitrated helper cell epitope) can be used to deliver new antigen vaccines, which can enhance the immune ability of cells.

Various DNA origami modifications facilitate imaging or optical imaging based on the localization of the DNA-origami structure in cells or animal tissue, which is also applicable to the diagnosis of cancer [113]. Du et al. [114] constructed a nanocarrier that integrated photoacoustic imaging and photothermal therapy (PTT) using triangular DNA origami carrying nanogold rods. Due to the strong enhanced permeability and retention effect of triangular DNA origami at the tumor site, gold nanorods (GNRs) with photoacoustic contrast and photothermal ablation properties are carried to the tumor tissue through intravenous injection, revealing the tumor position when irradiated with NIR light while also effectively killing the tumor and inhibiting its regeneration, thereby prolonging the lives of tumor-bearing mice and effectively integrating diagnosis and treatment. Li et al. [115] found that the DNA origami structure containing AS1411 aptamer can deliver DOX and indocyanine green to the tumor site in a targeted way, thus achieving chemotherapy. In addition, Wu et al. [116] developed a strategy to construct genetically encoded DNA origami, which can significantly up-regulate p53 protein in cells, thus achieving effective tumor treatment. Also He et al. [117] constructed a rectangular DNA origami nanostructure, which can carry a specific enzyme targeting tumor cells (NQO1), thus providing an effective strategy for precise cancer treatment.

Li et al. [11] rationally designed a promising immunofluorescence strategy that involves loading ruthenium complexes into cervical-cancer-targeted DNA cages. In this study, such cages were used to carry fluorescent ruthenium polypyridyl complex dyes. Apts-DNA@Ru was formed as an alternative to a traditional immunohistochemistry reagent and overcomes the disadvantages of the latter; it avoids the traditional multi-stage process, facilitates rapid pathological detection within 1–2 h following incubation, and realizes the rapid and inexpensive diagnosis of clinical cervical cancer tumor tissue. Therefore, it has significant clinical-pathological-grading and surgical-judgment applications potential.

Protein-based drugs, such as insulin, enzymes, antibodies, and cytokines, among others, are often used to treat diseases (Figure 6a and c). However, protein drug molecules are easily degraded by enzymes in vivo and enter cells with difficulty [118,119]. Functional DNA nanostructures can be designed as carrier platforms for efficient protein delivery [120]. In particular, DNA-origami technology can be used to construct a hexagonal cylindrical nanorobot and load the antibody that arrests cell growth. The nanorobot can be “locked” by a nucleic acid aptamer that recognizes specific cells. The “lock” (nucleic acid aptamer) on the surface is opened when combined with the target cell to release the internal antibody [121]. DNA origami can also be used to prepare nanodrug-loaded structures that contain thrombin in the interior and nucleolin from the cancer-cell surface outside. When the thrombin-carrying DNA nanocarrier is injected intravenously and reaches the tumor site, the nucleolin combination opens the DNA-carrier structure and releases the thrombin, resulting in local coagulation in the tumor tissue. Consequently, the tumor tissue is “cut off,” which stagnates its growth and leads to gradual necrosis [122].

Figure 6

Applications of DNA origami in cancer therapy. Reprinted with permission from refs [126–128,136]. Copyright American Chemical Society and Wiley-VCH. (a) Protein-DNA interaction-based therapy. (b) DNA origami loaded with DOX for cancer therapy. (c) Synergistic therapy based on DNA origami. (d) DNA origami loaded with gold nanorods for photothermal therapy of tumors.

DOX, one of the most studied chemotherapeutic drugs, is a common anti-tumor small drug molecule; however, its use can lead to drug resistance in tumor cells, which limits its clinical use. DOX inhibits the growth of tumor cells by embedding DNA double strands and interfering with its normal function. Using DNA origami as a DOX-delivery carrier (Figure 6a and b) greatly improves its loading rate and targeting [123,124]. In one study (Figure 6a) [125,126], Samet developed cholesterol-modified DNA nanotubes that bind to and are absorbed by HeLa cervical cancer cells and induce cell death associated with caspase activation and higher membrane permeability. A new DNA nanostructure was designed as a DNA nanoscale precision guided missile (D-PGM) for efficiently loading chemotherapy drugs and accurately delivering them to specific target cells. D-PGM consists of two parts: a warhead and a guidance/control system. D-PGM, which is inherently biocompatible and degradable, is capable of accurately identifying target cancer cells in a complex biological environment and actively delivers the drug in a targeted manner [127].

In addition to therapeutic drugs (i.e., small molecules and proteins), functional nucleic acids (including siRNA, miRNA, short hairpin RNA (shRNA), antisense oligonucleotides, and nucleic acid aptamers, etc.) are also widely used to treat diseases (Figure 6a and c) [126,128,129]. Because a DNA-origami structure provides the same essential properties as nucleic acid drugs, it can be directly loaded by complementary base pairing. The CpG oligonucleotide sequence is an activator that induces an immune response and is regarded to be a signal that bacteria use when invading the mammalian immune system [130]. However, the natural CpG sequence is easily degraded by DNA endonuclease (DNase) in blood, and its TLR9 cell receptor usually exists in the cytoplasm rather than on the membrane. Therefore, developing a carrier with a high transmembrane-transport capacity that improves the stability of the CpG sequence in vivo is very important [131]. Presently, many DNA nanocarriers [132–134] are capable of multivalently carrying CpG sequences in the form of complementary base pairs, maintaining stability in serum and living cells for several hours, and efficiently delivering CpG to target cells and causing more-obvious immune responses, thereby providing potential immunotherapy opportunities while also revealing that DNA nanostructures are good carriers for immunotherapy applications.

RNA interference is greatly significant when treating many genetic diseases, and liposome vesicles are usually used as carriers to deliver siRNA to cells. As DNA nanocarriers accurately bind to siRNA through complementary base pairing, they have attracted increasing levels of attention. Presently, the DNA tetrahedron has been used to carry siRNA; it stabilizes the siRNA molecule, facilitates longer blood circulation, and silences specific genes in tumor cells, which is its purpose [135].

For example, Wu et al. [137] constructed an RNA/DNA-origami nanoplatform (RDO). By reasonably designing a series of short fiber-like antisense chains, the target mRNA is folded into a chemically well-defined hybrid RNA/DNA nucleic acid nanostructure with a high antisense-loading capacity. Cell-type-specific aptamers were precisely introduced into the RDO using hybridization technology. After internalization in the target cells, the mRNA RDO scaffold is cleaved by intracellular RNase H to release multiple short chains (antisense) and achieve targeted delivery. The released antisense chain recognizes the intracellular target mRNA at various binding sites and triggers effective gene silencing. The customized RDO-based nanoplatform was observed to significantly inhibit tumor-cell proliferation by silencing the PLK1 tumor-related gene. This platform can be used to effectively deliver gene therapy that efficiently and sensitively delivers and silences target genes in living cells.

Some noble-metal nanoparticles are candidates for the photoacoustic imaging and photothermal treatment of tumors due to their strong absorption and scattering characteristics in the NIR region, with GNRs as excellent representatives [138]. PTT is a new tumor treatment method; it generates heat that burns local tumor tissue by irradiating nanoparticles pre-injected into the tumor site with an infrared laser beam. The local temperature needs to be well controlled during treatment to avoid destroying healthy tissue around the tumor. Self-assembled DNA origami GNR complexes for bifunctional nanotherapy can be developed by modifying the dense short DNA strands on the surfaces of the GNRs and complementarily combining them with triangular DNA origami nanostructures, which enhances GNR uptake by MCF-7 cells and the photothermal ablation effect on tumor cells both in vitro and in vivo [130]. CO transport of multiple drug molecules enhances the therapeutic effect through synergy. Jiang et al. used triangular DNA origami as a carrier to carry DOX and p53 genes to construct a “nano-kite” (Figure 6d) that exhibited significantly improved inhibition efficiency for MCF-7/MDR cells in vitro and in vivo [136]. Molecular DOX, shRNA, and a nucleic acid aptamer that targets the MUC1 receptor can also be introduced using the same vector, which also exhibited good therapeutic effects toward MCF-7/MDR [25].

Additionally, Dass et al. [139] combines DNA origami technology and plasmonic sensing technology for highly sensitive detection of biomolecules. DNA origami is a technique that uses self-assembly of DNA molecules to form various complex structures, while plasmonic sensing utilizes the optical properties of metal nanostructures for molecular detection. By combining these two technologies, the researchers have developed a new type of biosensor that can detect single protein molecules.

Domljanovic et al. [140] present a dynamic DNA origami book biosensor that is precisely decorated with arrays of fluorophores acting as donors and acceptors and also fluorescence quenchers that produce a strong optical readout upon exposure to external stimuli for the single or dual detection of target oligonucleotides and miRNAs. This biosensor allowed the detection of target molecules either through the decrease of FRET or through an increase in the fluorescence intensity profile owing to a rotation of the constituent top layer of the structure. Single-DNA origami experiments showed that detection of two targets can be achieved simultaneously within 10 min with a limit of detection in the range of 1–10 pM. Therefore, DNA nanotechnology-based devices are particularly advantageous for applications in oncology, owing to being ideally suited for the detection of cancer-associated nucleic acids, including circulating tumor-derived DNA fragments (ctDNAs), circulating microRNAs (miRNAs), and other RNA species. Besides, Han et al. [141] developed a facile, label-free, and amplification-free electrochemical biosensor to detect miRNA by using DNA origami nanostructure-supported DNA probes, with methylene blue serving as the hybridization redox indicator. The linear detection range of this biosensor was from 0.1 pM to 10.0 nM, with a lower detection limit of 79.8 fM. The selectivity of the miRNA biosensor was also studied by observing the discrimination ability of single-base mismatched sequences. Because of the larger surface area and unprecedented customizability of DNA origami nanostructures, this strategy demonstrated great potential for sensitive, selective, and label-free determination of miRNA for translational biomedical research and clinical applications.

3.4 Other nucleic acid nanomaterials for cancer diagnosis and treatment

Many DNA nanostructures with various shapes and sizes, including DNA tetrahedrons, cubes, octahedrons, dodecahedrons, icosahedrons, triangular biconical structures, prismatic structures, bucky-spheres, DNA boxes, and DNA spheres can be constructed and applied using the branched-DNA construction methods presented above. These different nanocage structures vary in size, from about ten nanometers to hundreds of nanometers, with molecular weights as high as 60 MDa. Their structures are plastic and stimulus-responsive, and they are widely used to encapsulate and release proteins, polypeptides, nanoparticles, and drugs in a controlled manner [142]. DNA nanobelts [143], nanoflowers (Figure 7a) [144], and nanosponges (Figure 7b) [145] are also common DNA nanostructures.

Figure 7

(a) DNA nanoflowers and (b) DNA nanosponges. Reprinted with permission from refs [144,145]. Copyright (2017) Wiley-VCH and (2019) American Chemical Society.

Presently, researchers screen-out nucleic acid aptamers with high affinities for various targets, such as metal ions, small molecules, proteins, living cells, viruses, and bacteria [146–149]. A linear or hybridized nucleic acid aptamer forms a special secondary structure that closely binds to the target molecule when it contacts it. In addition, the development of systematic evolution of ligands by exponential enrichment (SELEX) technology and increasing levels of investment in the nucleic acid aptamer screening fields has continuously expanded the list of nucleic acid aptamer targets. Compared to antibodies, nucleic acid aptamers have many advantages, including smallness, ease of synthesis and modification, lack of toxicity, low immunogenicity, and the ability to reversibly denature; hence, nucleic acid aptamers are ideal candidates for use in systems that recognize various targets.

However, the use of pure nucleic acid nanomaterials and other inorganic or organic materials is associated with many limitations. Therefore, nucleic acid aptamers and DNA hydrogels are used to prepare composite materials in many studies. In addition, other DNA composite materials are also gradually being used in cancer diagnosis and treatment applications. For example, DNA nanogold-rod [10,87], calcium phosphate crystal [150], nanodiamond [151], polymer [152–154], and lipid [155,156] composites, among others, provide structural and functional diversification opportunities for DNA nanomaterials, thereby enhancing their applications’ advantages. Constructing complexes that combine different materials is a new direction for the development of DNA nanomaterials.

Animal teeth and bones are mainly composed of inorganic calcium phosphate, which is a natural biological mineral. The design and synthesis of calcium phosphate nanostructures have attracted extensive attention; however, the fine synthesis of calcium phosphate nanostructures faces many difficulties. Wu et al. [157] also realized precise control during the uniform growth of thin-layer calcium phosphate nanocluster DNA-origami surfaces using a particle-adhesion crystallization strategy. The formed mineralized calcium phosphate DNA-origami structure not only retains the fine structure of the original DNA template, but also exhibits significantly improved thermal stability and mechanical strength (Figure 8a), which broadens the applicability of DNA nanostructures and provides a new concept for the preparation of drug carriers with prescribed geometric structures and long-term biological activities. Moreover, the DNA/calcium-phosphate composite constructed by this strategy is modifiable, and the function of the streptavidin-modified surface of the DNA origami structure (or structures modified with other molecules) is not affected by mineralization [150].

Figure 8

Nucleic acid nanocomposites and their applications: (a) DNA-Ca-P composite materials; (b) DNA-polydopamine composite materials; (c) DNA-nanodiamond composite materials. Reprinted with permission from [152,157,163]. Copyright American Chemical Society and Wiley-VCH.

DNA origami structures provide methods for accurately spatially localizing fluorescent nanodiamonds (FNDs). Zhang et al. [151] constructed a reliable and widely applicable surface-modification strategy that uses a DNA-origami structure to disperse FNDs well and self-assemble them into predetermined geometries (Figure 8b). Optical studies showed that the fluorescence characteristics of the nitrogen-vacancy color centers in the FNDs are preserved during surface modification and DNA assembly. This composite component, which is based on a DNA nanostructure, provides an ideal platform for studying the electronic and optical properties of nanoparticles in relation to distance and direction [158], as well as the catalytic activities of biomolecular assemblies [159].

The surface-grafting strategy is a convenient method for synthesizing biomolecular polymer structures with preset characteristics that are characterized through the simple purification of conjugates and by generally high grafting densities [160,161]. While the atom transfer radical polymerization (ATRP) law facilitates polymerization under biologically relevant conditions [162], successful polymerization on the surface of a biomolecule requires the installation of initiators. Weil et al. [152–154] used a high-precision DNA-origami scaffold to anchor an ATRP initiator at a preset position, and then grew nanoscale-oriented polymers; they also loaded DNA enzyme (DNAzyme) to catalyze the polymerization of dopamine on the DNA origami structure in the presence of hydrogen peroxide (Figure 8c). Modifying photosensitive molecules in G-quadruplexes on DNA origami can promote dopamine polymerization when irradiated with visible light [28]. Light initiation does not require hydrogen peroxide, which simplifies the system, with polymerization regulated by controlling periods of light and dark. As polydopamine is highly stable and photothermally conversion-efficient, it can be used in photothermal tumor-treatment applications. Therefore, the DNA/polydopamine composite has broad application prospects in the nanomedicine field.

One factor limiting the application of DNA nanostructures is their reduced stability in the presence of DNA nucleases, low salt concentrations, high temperatures, and different pH values. In recent years, a variety of stabilization strategies have been developed. Examples include coating layers with polymers [164–166], silica [167], peptoids [168], as well as cross-linking [169,170], among others. In the future, it will be very important to study the performance of stable DNA origami assembled plasma sensors in the application environment.

Non-natural xeno nucleic acids (XNAs) provide additional chemical modifications to nucleotide backbones or nuclear bases and also provide valuable opportunities to extend the function of aptamer switches [171]. These polymers can produce materials with enhanced properties and new functions that are not available in natural DNA or RNA. Advances in polymerase engineering and solid-phase synthesis have enabled XNA sequences to evolve in vitro, and several studies have described skeleton-modified XNA aptamers that are not easily recognized by nucleases, thus demonstrating improved biological stability [172]. However, the biosafety of DNA nanostructures still needs to be evaluated on an ongoing basis because coating or modification on DNA nanostructures can have different effects. For applications in biosensors and therapeutics, high cost and long reaction times in synthesis are the most challenging obstacles to building large-scale complex DNA nanostructures. Raveendran et al. [173] report a biosensor platform using DNA origami featuring a central cavity with a target-specific DNA aptamer coupled with a nanopore read-out to enable individual biomarker detection. They show that the modulation of the ion current through the nanopore upon the DNA origami translocation strongly depends on the presence of the biomarker in the cavity. We exploit this to generate a biosensing platform with a limit of detection of 3 nM and capable of the detection of human C-reactive protein (CRP) in clinically relevant fluids. Kim et al. [174] proposed an electrochemical biosensor composed of Ag-inserted multifunctional DNA four-way junctions (MF-DNA 4WJ) and porous rhodium nanoparticles (pRhNPs) on an Au micro-gap electrode for CRP detection. The durability of the biosensor was improved by the introduction of pRhNPs that maintained the redox property for 10 days. In addition, eight Ag⁺ ions were inserted into the C–C mismatch of DNA-4WJs to increase the conductivity, thereby improving the sensitivity of the sensor. Also, the Au micro-gap electrode provides eight-time repeatability, which confirmed the reliability of CRP determination. An experiment to confirm the sensitivity and selectivity of the biosensor was conducted using human serum and a calibration curve was obtained. DNAzymes is one of the better materials for biosensors. The functionalities of DNAzymes depend on both the sequences of the DNAzymes and additional cofactors such as metal ions or proteins. Many chemical reactions including RNA cleavage, oxidative or hydrolytic DNA cleavage, DNA or RNA ligation, DNA phosphorylation, and peroxidase reactions based on G-quadruplexes have now been carried out by DNAzymes. Most of the DNAzymes can catalyze their respective reactions with multiple turnovers, which allows for sensitive detection when used as sensors [175]. In addition, Rutten et al. [176] designed an intelligent (i.e., assay specific) 3D DNA origami design method, to nanopattern the surface of encoded microparticles present in the channels of a continuous microfluidic platform (EvalutionTM). They use an “smart” design approach to achieve the desired improved overall biosensing performance. The combination of the assay-specific interreceptor distances together with an enhanced upward orientation of the aptamer bioreceptors immobilized on the DNA origami structures, leads to improved binding efficiency, kinetics, and signal intensity when compared to direct bioreceptor immobilization. This directly improves the performance of biosensors in terms of sensitivity, total signal strength, and signal-to-noise ratio while maintaining reproducibility.