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Progress in DNA Tetrahedral Nanomaterials and Their Functionalization Research
CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 47, Issue 11, November 2019 Online English edition of the Chinese language journal
Cite this article as: Chinese J. Anal. Chem., 2019, 47(11): 1742–1750
REVIEW
Progress in DNA Tetrahedral Nanomaterials and Their Functionalization Research YU Li-Xing1,2, ZHAI Rui2, GONG Xiao-Yun2, XIE Jie2, HUANG Ze-Jian2, LIU Mei-Ying2, JIANG You2, DAI Xin-Hua2, FANG Xiang2,*, YU Xiao-Ping1,* 1
College of Life Sciences, Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, China Jiliang University, Hangzhou 310018, China 2 Mass Spectrometry Engineering Technology Research Center, Center for Advanced Measurement Science, National Institute of Metrology, Beijing 100029, China
Abstract:
DNA tetrahedral nanomaterials have been widely used in the fields of biosensing, drug delivery, bioimaging and
separation analysis because of their high stability, good biocompatibility and easy modification. Through different designs, specific functional molecules can be modified at DNA tetrahedral vertices, DNA tetrahedral cage structures, DNA double helix structures or DNA tetrahedron arms. The advantages of the materials combine well with the specific functions of the active molecules. This paper reviewed the development of DNA nanotechnology, and introduced four different functional modifications of DNA tetrahedron nanomaterials and their research status. Key Words:
DNA nanotechnology; DNA tetrahedron; Functionalization; Review
1 Introduction DNA is a biological macromolecule composed of four deoxynucleotides that are present in the nucleus as a carrier of genetic information. In 1953, James Watson and Francis Crick first proposed the theory of the DNA double helix[1]. Since then, DNA and its high-accuracy base pairing capability have attracted the attention of researchers and DNA has gradually been applied in the fields of medicine, genetics and ecology. Nanomaterials have unique electrical, optical and magnetic properties, and have a wide range of applications in home appliances, pharmaceuticals, environment protection and textile industries. Since DNA can self-assemble according to the principle of complementary pairing of bases, and the spatial structure has high controllability and precision[2], it is easy to construct various forms of DNA nanomaterials and show many unique advantages as follows: (1) nanomaterials based on DNA composition can easily penetrate negatively
charged cell membranes[3]; (2) DNA nanomaterials have almost no cytotoxicity compared to other nanomaterials, which are generally cytotoxic[4]; (3) these materials are very resistant to ribozymes and have high stability; and (4) the functionalized modification sites of these kinds of materials are abundant[5], and thus biomolecules or fluorescent dyes can be modified according to different needs. At present, DNA nanotechnology using DNA as a basic component has attracted wide attention[6]. Raniolo et al[7] analyzed the cellular uptake mediated by two different internalization pathways using folate-functionalized DNA octahedral nanocages. Liu et al[8] designed DNA nanoprobes for real-time imaging of cells and analyzed changes in Ca2+ and pH in mitochondria. Zhu et al[9] constructed a dynamic three-dimensional DNA nanopump that reversibly transports water and ferricyanide by binding pH-sensitive i-motif sequences at the edge of DNA nanostructures. Man et al[10] combined DNA nanotechnology with positive-feedback chemical reactions to achieve
________________________ Received 10 May 2019; accepted 4 July 2019 *Corresponding author. Email: [email protected]; [email protected] This work was supported by the National Key Research and Development Program of China (No. 2018YFF0212503) and the National Natural Science Foundation of China (No. 21575132). Copyright © 2019, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(19)61198-9
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self-assembly of active chiral plasmonic metamolecules. As a more classic and simplest polyhedron, DNA tetrahedral nanomaterials can be synthesized by simple self-assembly methods. At present, some review articles have also introduced DNA tetrahedral nanomaterials and their application progress. For example, Xie et al[11] reviewed and summarized the development and application of DNA tetrahedral nanomaterials in living cell research from two aspects of biosensors and drug delivery; Wang et al[12] reviewed current status of biological applications of three types DNA nanostructures (1D, 2D and 3D) in disease diagnosis and treatment; Su et al[13] described the latest research progress in the application of frame nucleic acid (FNA)-based biosensors in electrochemical detection, optical detection and intracellular sensing. To better utilize the advantages of DNA tetrahedral nanomaterials, it is often necessary to combine DNA tetrahedral nanomaterials with specific functional molecules. This paper reviews the different functionalization methods and applications of DNA tetrahedral nanomaterials.
2 Development of DNA nanotechnology and its synthesis and characterization 2.1
preparation of nanostructures of a target size by various etching techniques such as photolithography, grinding, and etching from a large-sized material (from micron to centimetre). This method is collectively referred to as nanoimprinting technology. The principle is that in the process of preparation, a single nanoconstruction unit is placed at a specific pattern position, and materials of various target shapes can be conveniently prepared by related techniques [23] such as scanning tunnelling-based imprinting techniques[24] and atomic force microscopy-based imprinting techniques[25]. The “bottom-up” approach refers to the passage of some smaller, simple structural units (such as atoms, molecules, nanoparticles, etc.) self-assemble by interaction forces to form relatively large and complex nanoscale structures[26], without manual intervention. With the rapid development of DNA nanotechnology in recent years, researchers have synthesized DNA nanomaterials of various shapes and sizes by adopting a “bottom-up” self-assembly method. As a more classic and simple polyhedron, DNA tetrahedrons can be synthesized by a simple self-assembly method. DNA tetrahedrons have a strong stability, low cytotoxicity, easy modification of structural sites, etc. They have been widely used as biosensors[27,28], gene carriers[29], drug delivery tools[30,31] and in other fields in recent years.
Development of DNA nanotechnology 2.2
In 1982, Professor Seeman[14] of New York University published the concept of “structural nanotechnology”, proposing that the forked Holiday structure was different from the conventional linear double-stranded DNA. In 1983, the team designed the Holiday structure and called the structure “Immobile Nucleic Acid Junctions”[15]. In 1993, Fu et al[16] improved the structure of the “Immobile Nucleic Acid Junctions” to form a more stable Double Crossover (DX) structure. In 1998, Winfree et al[17] first used the DX as a primitive structure to assemble a two-dimensional planar grid structure and confirmed the assembly with an atomic force microscope. In 2006, Rothemund et al[18] developed a new DNA self-assembly technique called DNA Origami. Through the use of this technique, Qian et al[19] designed a variety of DNA nanostructures such as triangles, rectangles, five-pointed stars and smiley faces, as well as the assembly of asymmetric maps of China. Andersen et al[20] designed a “dolphin” shape with a tail that could swing by applying it to the dynamic structure. Jia et al[21] proposed the concept of “DNA origami nanoreactor” and conducted further research on the regulation and assembly of DNA origami. In 2017, Tikhomirov et al[22] obtained a size-controlled DNA nanostructure by layered assembly, realizing the self-assembly of large-size DNA origami with different patterns. Depending on the nature and use of the material, nanostructures usually have two assembly methods: top-down and bottom-up. The “top-down” method refers to the
Synthesis and characterization of DNA tetrahedral nanostructures
The DNA tetrahedron is composed of four single-stranded DNAs that are paired with each other. Usually, the base sequence of four single-stranded DNAs is designed and then the number of bases per single-stranded DNA is divided into three small fragments, one small fragment of each single strand and one small fragment of another single strand complementary to each other to form one side of the DNA tetrahedron (Fig.1). The 5'- and 3'-end of each single-stranded DNA meet at the apex of the tetrahedron or form a port on the side. When merging at the apex, the specific functional molecule can be modified at the 5'- or 3'-end of the DNA single strand to realize functionalization of the DNA tetrahedron; when merging at the port, the port can be connected using DNA ligase or it can be modified with functional molecules[32]. At the same time, to make the two sides adjacent to the formed DNA tetrahedron have a certain angle, the DNA tetrahedral structure can be correctly formed and has a certain stability, and each of the two small fragments adjacent to each single-stranded DNA contains one or two bases that are not paired with any other sequence[33]. According to the principle of base pairing, the four singlestranded DNAs are added to the buffer in equal amounts. Through one-step annealing, the four single strands can automatically complement each other to form a threedimensional structure of DNA with a tetrahedral shape[34,35].
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Fig.1 Schematic diagram of self-assembly of DNA tetrahedral structure
Due to the obvious change in the structure and number of bases of DNA tetrahedrons, polyacrylamide gel electrophoresis can separate DNA that differs by several or tens of bases[36]. In experiments with polyacrylamide gel electrophoresis, DNA tetrahedrons move at a slower rate than other structures, so they can be characterized by the corresponding strip position of the sample [37]. High resolution transmission electron microscope (HRTEM) can also characterize the morphology of a DNA tetrahedron [35]. In addition, the synthetic DNA tetrahedron can also be observed by atomic force microscopy[38,39].
3 Functionalization of DNA tetrahedrons and their application Based on the different positions of the functional groups or molecules in the modification of DNA tetrahedrons, the modification methods can be mainly divided into the following four types: vertex type, capsule type, mosaic type and cantilever type[40] (Fig.2). Functionalized DNA tetrahedras have a wide range of applications in biosensing, drug delivery and bioimaging because of their combination of DNA tetrahedrons and specific functional molecules. 3.1
Application of vertex-modified DNA tetrahedral nanostructures
Vertex-type modification refers to the binding of functional molecular modifications to the apex of a DNA tetrahedron [40], such as modifying the three vertices of a DNA tetrahedron to a thiol group. The “Au‒S” bond and the DNA tetrahedron have a stable three-dimensional structure of a pyramid, and thus the DNA tetrahedral nanomaterial can then be stably fixed onto a
Fig.2
gold surface[41,44]. In addition, bioactive molecules or specific sequences for molecular recognition can be modified at the apex of DNA tetrahedrons according to experimental needs [45]. When synthesizing such modified DNA tetrahedral structures, it is often necessary to design a single-stranded DNA sequence to include a modified biologically active molecule or specific sequence at the 5'- or 3'-end of the singlestranded DNA. The two ends of the four single-stranded DNA converge at the vertices of the tetrahedron, and then the designed four single-stranded DNAs are added to the buffer in equal amounts and the hybridization is conducted to form a DNA tetrahedron by one-step annealing. MicroRNAs (miRNAs) are biomarkers for the diagnosis of early gastric cancer, and their sensitive detection is helpful for the early diagnosis, treatment and prognostic evaluation of gastric cancer. Qu et al[46] designed and developed a framework nucleic acid-based microarray for the analysis of multiple miRNAs. The probe was immobilized on the inner surface of an aldehyde-modified glass capillary by the amino group at the apex of the framework nucleic acid, and then the miRNAs to be assayed were added to the glass capillary to bind to the immobilized FNA probe. Finally, the fluorescence signal was enhanced by hybridization chain reaction (HCR) to achieve low concentration miRNA detection (100 fM) (Fig.3). Similar to microarrays, Qu et al[47] designed an aptamermodified DNA nanomaterial for the analysis of targets in nanolitre or picolitre volumetric droplets. In addition, Qu et al[48] designed a novel DNA nanostructured microarray (DNM) that could sensitively engage in fast selective detection of multiple heavy metal ions through DNM. Wang et al[49] hybridized a modified single-stranded DNA to form six sides of a DNA tetrahedron such that three amino groups and one side chain probe were located at the apex of the DNA tetrahedron. The DNA tetrahedron was immobilized on a glass slide by a reaction between an amino group and an epoxy group. Then, they used the overhanging probe to capture the target DNA sequence, allowing the capture probe, the target DNA sequence, and the biotin-modified detection probe to form a “sandwich” structure. Finally, the biotin was bound to the streptavidin-modified quantum dots, and the fluorescent spots obtained by epifluorescence microscopy were used for DNA counting. Metal complex drugs play an important role in the clinical
Four kinds of functional modifications of DNA tetrahedra: (A) Vertex[41]; (B) capsule[42]; (C) mosaic[43]; and (D) cantilever[29]
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could more easily break through the bottleneck of metal complex drugs that was difficult to diffuse into the nucleus, providing a new perspective for efficient and accurate glioma treatment. 3.2
Fig.3
Hybridization chain reaction (HCR) amplification based on framework nucleic acid (FNA) microarray for simultaneous detection of miRNAs[46]
treatment of cancer because of their ability to fight cancer in the nucleus. However, due to the barrier function of the nuclear membrane, only 1%–5% of the drugs in the cytoplasm can diffuse into the nucleus[50]. To increase the dose of drugs in the nucleus, DNA origami, which is biocompatible and easy to modify, has received wide attention from researchers. To avoid DNA denaturation caused by chemical modification, Tian et al[51] used a one-step method to link mucin 1 (MUC1) and aptamer AS1411 to the 5'-end of a DNA single strand. An anticancer metal complex ([Ir(ppy)2phen] + PF6) transport material (Apts-DNA@Ir) based on a double-targeted DNA tetrahedron was prepared (Fig.4). The results showed that when the material was incubated with the human glioma cell line U251 for 4 h, Apts-DNA@Ir gradually entered the nucleus while the free IrPP had difficulty in entering the nucleus. This dual-targeted design of DNA nanomaterials
Fig.4
Fig.5
Application of capsule-modified DNA tetrahedral nanostructures
Capsule-type modification refers to encapsulation of functionally modified molecules in a cage structure inside a DNA tetrahedron[40]. Li et al[52] proposed a method for packing gold nanoparticle clusters by encapsulating gold nanoparticles (AuNP) inside a DNA tetrahedral cage structure. As shown in Fig.5, they first modified the two thiol-modified single-stranded DNAs (green and blue) onto AuNPin and then hybridized them with the single-stranded (red) end of the DNA tetrahedron (TET) by a green DNA single strand. AuNPin was encapsulated in a DNA tetrahedron (AuNPin@TET). Then, another set of AuNPout was modified with a single strand (grey) of thiolated DNA that was complementary to the blue DNA single strand, and a novel DNA tetrahedral nanomaterial was formed by hybridizing a single strand of grey DNA with a single strand of blue DNA. One AuNP was in the material cage and four AuNPs were located at the centre of each face of the tetrahedron (similar to the structure of methane, Fig.5). This work prepared a new type of gold nanoparticle cluster with a similar methane structure, which provided a new approach to the preparation of metal nanoclusters. Some nanometre-sized materials can be encapsulated in the tetrahedron of DNA by using the cavity in the centre of the DNA tetrahedron. For example, a DNA tetrahedron coated with cytochrome C can regulate the entry of apoptotic enzyme
Schematic diagram of fabrication of Apts-DNA@Ir[51]
Self-assembly of methane-like nanoparticle-molecules[52]: (A) schematic diagram of AuNPin (AuNPout)4 assembly; (B) a methane molecule (AuNPs: gold nanoparticles)
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activator (Apaf-1). When Apaf-1 forms a complex with cytochrome C, it initiates an apoptotic protease cascade[53]. Erben et al[42] self-assembled DNA tetrahedrons, of which the central cavity could accommodate spheres with a radius of approximately 2.6 nm. They bound cytochrome C to the 5'-end of an oligonucleotide. The position of cytochrome C relative to the DNA tetrahedron (internal or external) could be regulated by altering the sequence of the oligonucleotide. This design may be applied to the regulation of protein function. Zhou et al[54] used DNA tetrahedrons coated with nanogold particles as monomers to prepare larger-sized DNA tetrahedral dendritic macromolecules. By replacing the gold nanoparticles with the corresponding antigens, this DNA tetrahedral dendrimer-gold nanoparticle conjugate method has a good possible application in cancer treatment[55] and [56] immunotherapy . 3.3 Application of mosaic-modified DNA tetrahedral nanostructures Mosaic modification refers to the incorporation of functionalized molecules or groups into the interior of a DNA tetrahedral double helix by conjugation[40]. For bioimaging analysis or analysis of DNA tetrahedral nanomaterial delivery pathways, fluorescently labelled biomolecules or dyes are often embedded in the interior of a DNA tetrahedral double helix using mosaic-type functionalization methods[57,58]. Mosaic-type modifications are also widely used in the delivery of anticancer drugs[60]. The anticancer drug is embedded in the side of the DNA tetrahedron, which can then independently pass through the negatively charged cell membrane and has almost no cytotoxicity, so that the maximum amount of the anticancer drug can be brought into the target cell, thereby effectively improving the drug utilization rate. This approach can also greatly reduce its adverse systemic effects on the human body. Free doxorubicin (Dox) enters the target cells in only a small amount and has almost no cytotoxicity against drugresistant cells[59]. Dox bound to DNA tetrahedral nanomaterials can enter the target cells in a much larger amount, which is more toxic to drug-resistant cells. The use of DNA tetrahedra for the delivery of Dox into drug-resistant breast cancer cells can better overcome the problem of target cell resistance. Kim et al[60] incubated Dox with a DNA tetrahedron (Td) (Dox@Td) to embed Dox inside the DNA tetrahedral double helix structure, and then the unloaded Dox was removed by G25 gel filtration. An estimated 26 Dox molecules were loaded into each Td. The experimental results showed that Dox@Td as a drug delivery system could significantly inhibit the growth of drug-resistant cells. It has a good application prospect in clinical use as a drug transport carrier for overcoming the drug resistance of cancer cells. MUC1 is overexpressed in most adenocarcinomas and it
has become an important molecular target for cancer therapy[61–63]. Dai et al[64] combined an aptamer (Apt) that could recognize MUC1 with a DNA tetrahedron (Td) that could carry Dox to establish a targeted drug delivery system (Apt-Td-Dox) (Fig.6). Drug loading experiments showed that each Apt-Td complex could carry approximately 25 Dox molecules and thus the maximum density of Dox could be delivered to MUC1-positive breast cancer cells using the characteristics of the DNA tetrahedrons. Apt-Td loaded with Dox showed higher cytotoxicity against MUC1-positive cancer cells compared to MUC1 negative control cells in vitro (p < 0.01). Therefore, they suggested that Apt-Td may become a carrier for effective drugs for the treatment of breast cancer with MUC1 expression. On the basis of functionalized nanomaterials with targeted drug delivery capabilities, Liu et al[65] achieved imaging analysis of MUC1 protein on cell membranes by adding probes with fluorescent properties to nanocarrier materials to distinguish MUC1 positive cells and MUC1 negative cells. They constructed a nanocarrier material (MUC1-Td-AS1411) consisting of a Dox-loaded DNA tetrahedron (Td), a MUC1 aptamer probe and an AS1411 aptamer. When the AS1411 aptamer selectively bound to the nucleolar protein, the nanocarrier material entered the nucleus and released Dox into the nucleus. Compared with free Dox, Dox loaded on MUC1-Td-AS1411 showed lower cytotoxicity to MUC1negative HL-7702 cells (p < 0.01), had approximately the same effect on sensitive MCF-7 cells (p > 0.05) and showed a better killing effect against adriamycin-resistant MCF-7 cells (p < 0.01). Li et al[66] combined CdTe quantum dots (QDs) as signal indicators with DNA tetrahedron (DNA TET) by hybridization,
Fig.6
Schematic of aptamer-modified DNA tetrahedron (Td) for selective delivery of doxorubicin (Dox) to mucin 1 (MUC1)positive breast cancer cells[64]
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then a large amount of methylene blue (MB) was inserted as a signal enhancer into the edge double strand of the DNA tetrahedron, and a photoelectrochemical (PEC) biosensor with near zero background noise was established (Fig.7). The sensor had a wide detection range of 50 aM–50 pM for microRNA-141 (miRNA-141) and a detection limit of 17 aM. To detect human IgG with high sensitivity, Ding et al[67] first immobilized streptavidin-conjugated anti-human IgG antibody (SA-Ab) on the surface of silanized glass plates by an antigen (Ag). Then, the biotin-containing DNA tetrahedron was attached to the SA-Ab by the specific reaction of biotin and streptavidin, and finally the fluorescent dye SYBR Green I was inserted into the DNA tetrahedral nanostructure. Fluorescence imaging analysis of the material was performed using an epifluorescence microscope and an electron multiplying charge coupler (Fig.8). The number of human IgG molecules was calculated by measuring the fluorescent spots one by one. Further experiments showed that when the concentration of human IgG ranged from 3.0 × 10–14 M to 1.0 × 10–12 M, the number of fluorescent spots on the image showed a good linear relationship with the concentration of human IgG.
TET: tetrahedron; MB: methylene blue; QDs: quantum dots
3.4 Application of cantilever-modified DNA tetrahedral nanostructures
Fig.8
Cantilever-type modification refers to suspending a functionalized molecule or group on the side arm of a DNA tetrahedral structure[40]. For example, by designing the base sequences of four single-stranded DNAs, the 5'- and 3'-end junctions of single-stranded DNA are on the side of the DNA tetrahedral nanostructure (middle or other non-vertex) and the 5'- or 3'-end that reveal no complementary pairing extends outward for their modification by functional molecules [29,31]. Telomerase activity is tightly regulated in normal cells, but in most malignant cells, its activity is increased and it may be involved in the malignant transformation of tumour cells[68,69]. Therefore, monitoring of telomerase activity is important for the diagnosis and treatment of cancer. To avoid the use of current probes to measure intracellular telomerase activity, which was interfered with by false positive results, Meng et al[70] proposed a new fluorescence resonance energy transfer (FRET) method for monitoring telomerase activity in cells. They designed DNA nanoprobes constructed from DNA tetrahedrons and Flare DNA. Since active telomerase could catalyze DNA extension, altering the structure of the DNA nanoprobe resulted in an increase in the distance between the two fluorophores of the label, thereby significantly reducing the efficiency of FRET (Fig.9). The ratio sensors prepared by this method can be used for the detection of telomerase activity at the single cell level. In addition, the method can be further used for the detection of telomerase inhibitors, which helps to discover new anticancer drugs.
Fig.7
Preparation of DNA TET-CdTe QDs-MB complex[66]
Detection of human immunoglobulin G (IgG) by DNA tetrahedral nanocomposites[67]
Ag, antigen; BSA, bovine serum albumin; Bio-DT, bio-DNA tetrahedron; SA-Ab, strepavidin-antibody
Fig.9 Schematic diagram of monitoring telomerase activity in cells[70]
Lee et al[29] self-assembled a DNA tetrahedron of a defined size that could deliver siRNAs into cells to silence tumour-associated target genes. They first designed six DNA single strands with complementary overhangs at the 3'-end and then obtained DNA tetrahedral nanomaterials by selfassembly in which unpaired sequences were built into each side of the tetrahedron for ligation of siRNA sequences. Finally, the siRNA entered the interior of the cell by immobilization on the DNA tetrahedron. Charoenphol et al[31] designed DNA tetrahedral
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nanomaterials into a plurality of overhangs as sites that binding to a targeting ligand. Previous studies confirmed that the nucleic acid aptamer AS1411 had an inhibitory effect on tumour cell proliferation[71,72], so AS1411 was attached to the overhangs of the DNA tetrahedron. The experimental results showed that DNA tetrahedral nanomaterial modified with AS1411 could inhibit the growth of HeLa cells within 24 h, and its high selectivity had almost no adverse effects on the growth of non-cancer cell lines. This method provided a reference for the delivery of a plurality of biologically active molecules based on DNA tetrahedrons as a vehicle for synergistic treatment.
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4 Summary and outlook
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Functionalized DNA tetrahedral nanomaterials have many advantages including simple self-assembly, stable mechanical properties, superior biocompatibility and stability in the presence of ribozymes. They have good application prospects in biosensing, separation analysis, bioimaging and drug delivery. Functionalized DNA tetrahedral nanomaterials are easily constructed into specifically enriched materials. Through the combination of materials and target substances, the separation and qualitative analysis of target substances in complex systems are realized. However, functionalized DNA tetrahedral nanomaterials have some difficulties in quantitative analysis of target substances due to the inability to ascertain the exact amount of material captured by the target material. In the field of biological diagnosis and treatment, related in vivo experiments with DNA tetrahedral nanomaterials still mainly use mice as experimental subjects, and they still face many challenges to overcome before carrying out related experiments in the human body. DNA tetrahedral nanomaterials enter the cell and then enter the lysosome[73]. However, it is still unclear whether this would ultimately affect their use. In addition, it still needs further study whether the biologically active molecules, fluorescent dyes and probes used to functionalize modified DNA tetrahedral nanomaterials have adverse effects after entering the body.
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Cite this article as: Chinese J. Anal. Chem., 2019, 47(11): 1742–1750
REVIEW
Progress in DNA Tetrahedral Nanomaterials and Their Functionalization Research YU Li-Xing1,2, ZHAI Rui2, GONG Xiao-Yun2, XIE Jie2, HUANG Ze-Jian2, LIU Mei-Ying2, JIANG You2, DAI Xin-Hua2, FANG Xiang2,*, YU Xiao-Ping1,* 1
College of Life Sciences, Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, China Jiliang University, Hangzhou 310018, China 2 Mass Spectrometry Engineering Technology Research Center, Center for Advanced Measurement Science, National Institute of Metrology, Beijing 100029, China
Abstract:
DNA tetrahedral nanomaterials have been widely used in the fields of biosensing, drug delivery, bioimaging and
separation analysis because of their high stability, good biocompatibility and easy modification. Through different designs, specific functional molecules can be modified at DNA tetrahedral vertices, DNA tetrahedral cage structures, DNA double helix structures or DNA tetrahedron arms. The advantages of the materials combine well with the specific functions of the active molecules. This paper reviewed the development of DNA nanotechnology, and introduced four different functional modifications of DNA tetrahedron nanomaterials and their research status. Key Words:
DNA nanotechnology; DNA tetrahedron; Functionalization; Review
1 Introduction DNA is a biological macromolecule composed of four deoxynucleotides that are present in the nucleus as a carrier of genetic information. In 1953, James Watson and Francis Crick first proposed the theory of the DNA double helix[1]. Since then, DNA and its high-accuracy base pairing capability have attracted the attention of researchers and DNA has gradually been applied in the fields of medicine, genetics and ecology. Nanomaterials have unique electrical, optical and magnetic properties, and have a wide range of applications in home appliances, pharmaceuticals, environment protection and textile industries. Since DNA can self-assemble according to the principle of complementary pairing of bases, and the spatial structure has high controllability and precision[2], it is easy to construct various forms of DNA nanomaterials and show many unique advantages as follows: (1) nanomaterials based on DNA composition can easily penetrate negatively
charged cell membranes[3]; (2) DNA nanomaterials have almost no cytotoxicity compared to other nanomaterials, which are generally cytotoxic[4]; (3) these materials are very resistant to ribozymes and have high stability; and (4) the functionalized modification sites of these kinds of materials are abundant[5], and thus biomolecules or fluorescent dyes can be modified according to different needs. At present, DNA nanotechnology using DNA as a basic component has attracted wide attention[6]. Raniolo et al[7] analyzed the cellular uptake mediated by two different internalization pathways using folate-functionalized DNA octahedral nanocages. Liu et al[8] designed DNA nanoprobes for real-time imaging of cells and analyzed changes in Ca2+ and pH in mitochondria. Zhu et al[9] constructed a dynamic three-dimensional DNA nanopump that reversibly transports water and ferricyanide by binding pH-sensitive i-motif sequences at the edge of DNA nanostructures. Man et al[10] combined DNA nanotechnology with positive-feedback chemical reactions to achieve
________________________ Received 10 May 2019; accepted 4 July 2019 *Corresponding author. Email: [email protected]; [email protected] This work was supported by the National Key Research and Development Program of China (No. 2018YFF0212503) and the National Natural Science Foundation of China (No. 21575132). Copyright © 2019, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(19)61198-9
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self-assembly of active chiral plasmonic metamolecules. As a more classic and simplest polyhedron, DNA tetrahedral nanomaterials can be synthesized by simple self-assembly methods. At present, some review articles have also introduced DNA tetrahedral nanomaterials and their application progress. For example, Xie et al[11] reviewed and summarized the development and application of DNA tetrahedral nanomaterials in living cell research from two aspects of biosensors and drug delivery; Wang et al[12] reviewed current status of biological applications of three types DNA nanostructures (1D, 2D and 3D) in disease diagnosis and treatment; Su et al[13] described the latest research progress in the application of frame nucleic acid (FNA)-based biosensors in electrochemical detection, optical detection and intracellular sensing. To better utilize the advantages of DNA tetrahedral nanomaterials, it is often necessary to combine DNA tetrahedral nanomaterials with specific functional molecules. This paper reviews the different functionalization methods and applications of DNA tetrahedral nanomaterials.
2 Development of DNA nanotechnology and its synthesis and characterization 2.1
preparation of nanostructures of a target size by various etching techniques such as photolithography, grinding, and etching from a large-sized material (from micron to centimetre). This method is collectively referred to as nanoimprinting technology. The principle is that in the process of preparation, a single nanoconstruction unit is placed at a specific pattern position, and materials of various target shapes can be conveniently prepared by related techniques [23] such as scanning tunnelling-based imprinting techniques[24] and atomic force microscopy-based imprinting techniques[25]. The “bottom-up” approach refers to the passage of some smaller, simple structural units (such as atoms, molecules, nanoparticles, etc.) self-assemble by interaction forces to form relatively large and complex nanoscale structures[26], without manual intervention. With the rapid development of DNA nanotechnology in recent years, researchers have synthesized DNA nanomaterials of various shapes and sizes by adopting a “bottom-up” self-assembly method. As a more classic and simple polyhedron, DNA tetrahedrons can be synthesized by a simple self-assembly method. DNA tetrahedrons have a strong stability, low cytotoxicity, easy modification of structural sites, etc. They have been widely used as biosensors[27,28], gene carriers[29], drug delivery tools[30,31] and in other fields in recent years.
Development of DNA nanotechnology 2.2
In 1982, Professor Seeman[14] of New York University published the concept of “structural nanotechnology”, proposing that the forked Holiday structure was different from the conventional linear double-stranded DNA. In 1983, the team designed the Holiday structure and called the structure “Immobile Nucleic Acid Junctions”[15]. In 1993, Fu et al[16] improved the structure of the “Immobile Nucleic Acid Junctions” to form a more stable Double Crossover (DX) structure. In 1998, Winfree et al[17] first used the DX as a primitive structure to assemble a two-dimensional planar grid structure and confirmed the assembly with an atomic force microscope. In 2006, Rothemund et al[18] developed a new DNA self-assembly technique called DNA Origami. Through the use of this technique, Qian et al[19] designed a variety of DNA nanostructures such as triangles, rectangles, five-pointed stars and smiley faces, as well as the assembly of asymmetric maps of China. Andersen et al[20] designed a “dolphin” shape with a tail that could swing by applying it to the dynamic structure. Jia et al[21] proposed the concept of “DNA origami nanoreactor” and conducted further research on the regulation and assembly of DNA origami. In 2017, Tikhomirov et al[22] obtained a size-controlled DNA nanostructure by layered assembly, realizing the self-assembly of large-size DNA origami with different patterns. Depending on the nature and use of the material, nanostructures usually have two assembly methods: top-down and bottom-up. The “top-down” method refers to the
Synthesis and characterization of DNA tetrahedral nanostructures
The DNA tetrahedron is composed of four single-stranded DNAs that are paired with each other. Usually, the base sequence of four single-stranded DNAs is designed and then the number of bases per single-stranded DNA is divided into three small fragments, one small fragment of each single strand and one small fragment of another single strand complementary to each other to form one side of the DNA tetrahedron (Fig.1). The 5'- and 3'-end of each single-stranded DNA meet at the apex of the tetrahedron or form a port on the side. When merging at the apex, the specific functional molecule can be modified at the 5'- or 3'-end of the DNA single strand to realize functionalization of the DNA tetrahedron; when merging at the port, the port can be connected using DNA ligase or it can be modified with functional molecules[32]. At the same time, to make the two sides adjacent to the formed DNA tetrahedron have a certain angle, the DNA tetrahedral structure can be correctly formed and has a certain stability, and each of the two small fragments adjacent to each single-stranded DNA contains one or two bases that are not paired with any other sequence[33]. According to the principle of base pairing, the four singlestranded DNAs are added to the buffer in equal amounts. Through one-step annealing, the four single strands can automatically complement each other to form a threedimensional structure of DNA with a tetrahedral shape[34,35].
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Fig.1 Schematic diagram of self-assembly of DNA tetrahedral structure
Due to the obvious change in the structure and number of bases of DNA tetrahedrons, polyacrylamide gel electrophoresis can separate DNA that differs by several or tens of bases[36]. In experiments with polyacrylamide gel electrophoresis, DNA tetrahedrons move at a slower rate than other structures, so they can be characterized by the corresponding strip position of the sample [37]. High resolution transmission electron microscope (HRTEM) can also characterize the morphology of a DNA tetrahedron [35]. In addition, the synthetic DNA tetrahedron can also be observed by atomic force microscopy[38,39].
3 Functionalization of DNA tetrahedrons and their application Based on the different positions of the functional groups or molecules in the modification of DNA tetrahedrons, the modification methods can be mainly divided into the following four types: vertex type, capsule type, mosaic type and cantilever type[40] (Fig.2). Functionalized DNA tetrahedras have a wide range of applications in biosensing, drug delivery and bioimaging because of their combination of DNA tetrahedrons and specific functional molecules. 3.1
Application of vertex-modified DNA tetrahedral nanostructures
Vertex-type modification refers to the binding of functional molecular modifications to the apex of a DNA tetrahedron [40], such as modifying the three vertices of a DNA tetrahedron to a thiol group. The “Au‒S” bond and the DNA tetrahedron have a stable three-dimensional structure of a pyramid, and thus the DNA tetrahedral nanomaterial can then be stably fixed onto a
Fig.2
gold surface[41,44]. In addition, bioactive molecules or specific sequences for molecular recognition can be modified at the apex of DNA tetrahedrons according to experimental needs [45]. When synthesizing such modified DNA tetrahedral structures, it is often necessary to design a single-stranded DNA sequence to include a modified biologically active molecule or specific sequence at the 5'- or 3'-end of the singlestranded DNA. The two ends of the four single-stranded DNA converge at the vertices of the tetrahedron, and then the designed four single-stranded DNAs are added to the buffer in equal amounts and the hybridization is conducted to form a DNA tetrahedron by one-step annealing. MicroRNAs (miRNAs) are biomarkers for the diagnosis of early gastric cancer, and their sensitive detection is helpful for the early diagnosis, treatment and prognostic evaluation of gastric cancer. Qu et al[46] designed and developed a framework nucleic acid-based microarray for the analysis of multiple miRNAs. The probe was immobilized on the inner surface of an aldehyde-modified glass capillary by the amino group at the apex of the framework nucleic acid, and then the miRNAs to be assayed were added to the glass capillary to bind to the immobilized FNA probe. Finally, the fluorescence signal was enhanced by hybridization chain reaction (HCR) to achieve low concentration miRNA detection (100 fM) (Fig.3). Similar to microarrays, Qu et al[47] designed an aptamermodified DNA nanomaterial for the analysis of targets in nanolitre or picolitre volumetric droplets. In addition, Qu et al[48] designed a novel DNA nanostructured microarray (DNM) that could sensitively engage in fast selective detection of multiple heavy metal ions through DNM. Wang et al[49] hybridized a modified single-stranded DNA to form six sides of a DNA tetrahedron such that three amino groups and one side chain probe were located at the apex of the DNA tetrahedron. The DNA tetrahedron was immobilized on a glass slide by a reaction between an amino group and an epoxy group. Then, they used the overhanging probe to capture the target DNA sequence, allowing the capture probe, the target DNA sequence, and the biotin-modified detection probe to form a “sandwich” structure. Finally, the biotin was bound to the streptavidin-modified quantum dots, and the fluorescent spots obtained by epifluorescence microscopy were used for DNA counting. Metal complex drugs play an important role in the clinical
Four kinds of functional modifications of DNA tetrahedra: (A) Vertex[41]; (B) capsule[42]; (C) mosaic[43]; and (D) cantilever[29]
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could more easily break through the bottleneck of metal complex drugs that was difficult to diffuse into the nucleus, providing a new perspective for efficient and accurate glioma treatment. 3.2
Fig.3
Hybridization chain reaction (HCR) amplification based on framework nucleic acid (FNA) microarray for simultaneous detection of miRNAs[46]
treatment of cancer because of their ability to fight cancer in the nucleus. However, due to the barrier function of the nuclear membrane, only 1%–5% of the drugs in the cytoplasm can diffuse into the nucleus[50]. To increase the dose of drugs in the nucleus, DNA origami, which is biocompatible and easy to modify, has received wide attention from researchers. To avoid DNA denaturation caused by chemical modification, Tian et al[51] used a one-step method to link mucin 1 (MUC1) and aptamer AS1411 to the 5'-end of a DNA single strand. An anticancer metal complex ([Ir(ppy)2phen] + PF6) transport material (Apts-DNA@Ir) based on a double-targeted DNA tetrahedron was prepared (Fig.4). The results showed that when the material was incubated with the human glioma cell line U251 for 4 h, Apts-DNA@Ir gradually entered the nucleus while the free IrPP had difficulty in entering the nucleus. This dual-targeted design of DNA nanomaterials
Fig.4
Fig.5
Application of capsule-modified DNA tetrahedral nanostructures
Capsule-type modification refers to encapsulation of functionally modified molecules in a cage structure inside a DNA tetrahedron[40]. Li et al[52] proposed a method for packing gold nanoparticle clusters by encapsulating gold nanoparticles (AuNP) inside a DNA tetrahedral cage structure. As shown in Fig.5, they first modified the two thiol-modified single-stranded DNAs (green and blue) onto AuNPin and then hybridized them with the single-stranded (red) end of the DNA tetrahedron (TET) by a green DNA single strand. AuNPin was encapsulated in a DNA tetrahedron (AuNPin@TET). Then, another set of AuNPout was modified with a single strand (grey) of thiolated DNA that was complementary to the blue DNA single strand, and a novel DNA tetrahedral nanomaterial was formed by hybridizing a single strand of grey DNA with a single strand of blue DNA. One AuNP was in the material cage and four AuNPs were located at the centre of each face of the tetrahedron (similar to the structure of methane, Fig.5). This work prepared a new type of gold nanoparticle cluster with a similar methane structure, which provided a new approach to the preparation of metal nanoclusters. Some nanometre-sized materials can be encapsulated in the tetrahedron of DNA by using the cavity in the centre of the DNA tetrahedron. For example, a DNA tetrahedron coated with cytochrome C can regulate the entry of apoptotic enzyme
Schematic diagram of fabrication of Apts-DNA@Ir[51]
Self-assembly of methane-like nanoparticle-molecules[52]: (A) schematic diagram of AuNPin (AuNPout)4 assembly; (B) a methane molecule (AuNPs: gold nanoparticles)
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activator (Apaf-1). When Apaf-1 forms a complex with cytochrome C, it initiates an apoptotic protease cascade[53]. Erben et al[42] self-assembled DNA tetrahedrons, of which the central cavity could accommodate spheres with a radius of approximately 2.6 nm. They bound cytochrome C to the 5'-end of an oligonucleotide. The position of cytochrome C relative to the DNA tetrahedron (internal or external) could be regulated by altering the sequence of the oligonucleotide. This design may be applied to the regulation of protein function. Zhou et al[54] used DNA tetrahedrons coated with nanogold particles as monomers to prepare larger-sized DNA tetrahedral dendritic macromolecules. By replacing the gold nanoparticles with the corresponding antigens, this DNA tetrahedral dendrimer-gold nanoparticle conjugate method has a good possible application in cancer treatment[55] and [56] immunotherapy . 3.3 Application of mosaic-modified DNA tetrahedral nanostructures Mosaic modification refers to the incorporation of functionalized molecules or groups into the interior of a DNA tetrahedral double helix by conjugation[40]. For bioimaging analysis or analysis of DNA tetrahedral nanomaterial delivery pathways, fluorescently labelled biomolecules or dyes are often embedded in the interior of a DNA tetrahedral double helix using mosaic-type functionalization methods[57,58]. Mosaic-type modifications are also widely used in the delivery of anticancer drugs[60]. The anticancer drug is embedded in the side of the DNA tetrahedron, which can then independently pass through the negatively charged cell membrane and has almost no cytotoxicity, so that the maximum amount of the anticancer drug can be brought into the target cell, thereby effectively improving the drug utilization rate. This approach can also greatly reduce its adverse systemic effects on the human body. Free doxorubicin (Dox) enters the target cells in only a small amount and has almost no cytotoxicity against drugresistant cells[59]. Dox bound to DNA tetrahedral nanomaterials can enter the target cells in a much larger amount, which is more toxic to drug-resistant cells. The use of DNA tetrahedra for the delivery of Dox into drug-resistant breast cancer cells can better overcome the problem of target cell resistance. Kim et al[60] incubated Dox with a DNA tetrahedron (Td) (Dox@Td) to embed Dox inside the DNA tetrahedral double helix structure, and then the unloaded Dox was removed by G25 gel filtration. An estimated 26 Dox molecules were loaded into each Td. The experimental results showed that Dox@Td as a drug delivery system could significantly inhibit the growth of drug-resistant cells. It has a good application prospect in clinical use as a drug transport carrier for overcoming the drug resistance of cancer cells. MUC1 is overexpressed in most adenocarcinomas and it
has become an important molecular target for cancer therapy[61–63]. Dai et al[64] combined an aptamer (Apt) that could recognize MUC1 with a DNA tetrahedron (Td) that could carry Dox to establish a targeted drug delivery system (Apt-Td-Dox) (Fig.6). Drug loading experiments showed that each Apt-Td complex could carry approximately 25 Dox molecules and thus the maximum density of Dox could be delivered to MUC1-positive breast cancer cells using the characteristics of the DNA tetrahedrons. Apt-Td loaded with Dox showed higher cytotoxicity against MUC1-positive cancer cells compared to MUC1 negative control cells in vitro (p < 0.01). Therefore, they suggested that Apt-Td may become a carrier for effective drugs for the treatment of breast cancer with MUC1 expression. On the basis of functionalized nanomaterials with targeted drug delivery capabilities, Liu et al[65] achieved imaging analysis of MUC1 protein on cell membranes by adding probes with fluorescent properties to nanocarrier materials to distinguish MUC1 positive cells and MUC1 negative cells. They constructed a nanocarrier material (MUC1-Td-AS1411) consisting of a Dox-loaded DNA tetrahedron (Td), a MUC1 aptamer probe and an AS1411 aptamer. When the AS1411 aptamer selectively bound to the nucleolar protein, the nanocarrier material entered the nucleus and released Dox into the nucleus. Compared with free Dox, Dox loaded on MUC1-Td-AS1411 showed lower cytotoxicity to MUC1negative HL-7702 cells (p < 0.01), had approximately the same effect on sensitive MCF-7 cells (p > 0.05) and showed a better killing effect against adriamycin-resistant MCF-7 cells (p < 0.01). Li et al[66] combined CdTe quantum dots (QDs) as signal indicators with DNA tetrahedron (DNA TET) by hybridization,
Fig.6
Schematic of aptamer-modified DNA tetrahedron (Td) for selective delivery of doxorubicin (Dox) to mucin 1 (MUC1)positive breast cancer cells[64]
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then a large amount of methylene blue (MB) was inserted as a signal enhancer into the edge double strand of the DNA tetrahedron, and a photoelectrochemical (PEC) biosensor with near zero background noise was established (Fig.7). The sensor had a wide detection range of 50 aM–50 pM for microRNA-141 (miRNA-141) and a detection limit of 17 aM. To detect human IgG with high sensitivity, Ding et al[67] first immobilized streptavidin-conjugated anti-human IgG antibody (SA-Ab) on the surface of silanized glass plates by an antigen (Ag). Then, the biotin-containing DNA tetrahedron was attached to the SA-Ab by the specific reaction of biotin and streptavidin, and finally the fluorescent dye SYBR Green I was inserted into the DNA tetrahedral nanostructure. Fluorescence imaging analysis of the material was performed using an epifluorescence microscope and an electron multiplying charge coupler (Fig.8). The number of human IgG molecules was calculated by measuring the fluorescent spots one by one. Further experiments showed that when the concentration of human IgG ranged from 3.0 × 10–14 M to 1.0 × 10–12 M, the number of fluorescent spots on the image showed a good linear relationship with the concentration of human IgG.
TET: tetrahedron; MB: methylene blue; QDs: quantum dots
3.4 Application of cantilever-modified DNA tetrahedral nanostructures
Fig.8
Cantilever-type modification refers to suspending a functionalized molecule or group on the side arm of a DNA tetrahedral structure[40]. For example, by designing the base sequences of four single-stranded DNAs, the 5'- and 3'-end junctions of single-stranded DNA are on the side of the DNA tetrahedral nanostructure (middle or other non-vertex) and the 5'- or 3'-end that reveal no complementary pairing extends outward for their modification by functional molecules [29,31]. Telomerase activity is tightly regulated in normal cells, but in most malignant cells, its activity is increased and it may be involved in the malignant transformation of tumour cells[68,69]. Therefore, monitoring of telomerase activity is important for the diagnosis and treatment of cancer. To avoid the use of current probes to measure intracellular telomerase activity, which was interfered with by false positive results, Meng et al[70] proposed a new fluorescence resonance energy transfer (FRET) method for monitoring telomerase activity in cells. They designed DNA nanoprobes constructed from DNA tetrahedrons and Flare DNA. Since active telomerase could catalyze DNA extension, altering the structure of the DNA nanoprobe resulted in an increase in the distance between the two fluorophores of the label, thereby significantly reducing the efficiency of FRET (Fig.9). The ratio sensors prepared by this method can be used for the detection of telomerase activity at the single cell level. In addition, the method can be further used for the detection of telomerase inhibitors, which helps to discover new anticancer drugs.
Fig.7
Preparation of DNA TET-CdTe QDs-MB complex[66]
Detection of human immunoglobulin G (IgG) by DNA tetrahedral nanocomposites[67]
Ag, antigen; BSA, bovine serum albumin; Bio-DT, bio-DNA tetrahedron; SA-Ab, strepavidin-antibody
Fig.9 Schematic diagram of monitoring telomerase activity in cells[70]
Lee et al[29] self-assembled a DNA tetrahedron of a defined size that could deliver siRNAs into cells to silence tumour-associated target genes. They first designed six DNA single strands with complementary overhangs at the 3'-end and then obtained DNA tetrahedral nanomaterials by selfassembly in which unpaired sequences were built into each side of the tetrahedron for ligation of siRNA sequences. Finally, the siRNA entered the interior of the cell by immobilization on the DNA tetrahedron. Charoenphol et al[31] designed DNA tetrahedral
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nanomaterials into a plurality of overhangs as sites that binding to a targeting ligand. Previous studies confirmed that the nucleic acid aptamer AS1411 had an inhibitory effect on tumour cell proliferation[71,72], so AS1411 was attached to the overhangs of the DNA tetrahedron. The experimental results showed that DNA tetrahedral nanomaterial modified with AS1411 could inhibit the growth of HeLa cells within 24 h, and its high selectivity had almost no adverse effects on the growth of non-cancer cell lines. This method provided a reference for the delivery of a plurality of biologically active molecules based on DNA tetrahedrons as a vehicle for synergistic treatment.
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4 Summary and outlook
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Functionalized DNA tetrahedral nanomaterials have many advantages including simple self-assembly, stable mechanical properties, superior biocompatibility and stability in the presence of ribozymes. They have good application prospects in biosensing, separation analysis, bioimaging and drug delivery. Functionalized DNA tetrahedral nanomaterials are easily constructed into specifically enriched materials. Through the combination of materials and target substances, the separation and qualitative analysis of target substances in complex systems are realized. However, functionalized DNA tetrahedral nanomaterials have some difficulties in quantitative analysis of target substances due to the inability to ascertain the exact amount of material captured by the target material. In the field of biological diagnosis and treatment, related in vivo experiments with DNA tetrahedral nanomaterials still mainly use mice as experimental subjects, and they still face many challenges to overcome before carrying out related experiments in the human body. DNA tetrahedral nanomaterials enter the cell and then enter the lysosome[73]. However, it is still unclear whether this would ultimately affect their use. In addition, it still needs further study whether the biologically active molecules, fluorescent dyes and probes used to functionalize modified DNA tetrahedral nanomaterials have adverse effects after entering the body.
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