The capsid lattice engages a bipartite NUP153 motif to mediate nuclear entry of HIV-1 cores.

Increasing evidence has suggested that the HIV-1 capsid enters the nucleus in a largely assembled, intact form. However, not much is known about how the cone-shaped capsid interacts with the nucleoporins (NUPs) in the nuclear pore for crossing the nuclear pore complex. Here, we elucidate how NUP153 binds HIV-1 capsid by engaging the assembled capsid protein (CA) lattice. A bipartite motif containing both canonical and noncanonical interaction modules was identified at the C-terminal tail region of NUP153. The canonical cargo-targeting phenylalanine-glycine (FG) motif engaged the CA hexamer. By contrast, a previously unidentified triple-arginine (RRR) motif in NUP153 targeted HIV-1 capsid at the CA tri-hexamer interface in the capsid. HIV-1 infection studies indicated that both FG- and RRR-motifs were important for the nuclear import of HIV-1 cores. Moreover, the presence of NUP153 stabilized tubular CA assemblies in vitro. Our results provide molecular-level mechanistic evidence that NUP153 contributes to the entry of the intact capsid into the nucleus.

Functionalized DNA-Origami-Protein Nanopores Generate Large Transmembrane Channels with Programmable Size-Selectivity.

The DNA-origami technique has enabled the engineering of transmembrane nanopores with programmable size and functionality, showing promise in building biosensors and synthetic cells. However, it remains challenging to build large (>10 nm), functionalizable nanopores that spontaneously perforate lipid membranes. Here, we take advantage of pneumolysin (PLY), a bacterial toxin that potently forms wide ring-like channels on cell membranes, to construct hybrid DNA-protein nanopores. This PLY-DNA-origami complex, in which a DNA-origami ring corrals up to 48 copies of PLY, targets the cholesterol-rich membranes of liposomes and red blood cells, readily forming uniformly sized pores with an average inner diameter of ∼22 nm. Such hybrid nanopores facilitate the exchange of macromolecules between perforated liposomes and their environment, with the exchange rate negatively correlating with the macromolecule size (diameters of gyration: 8-22 nm). Additionally, the DNA ring can be decorated with intrinsically disordered nucleoporins to further restrict the diffusion of traversing molecules, highlighting the programmability of the hybrid nanopores. PLY-DNA pores provide an enabling biophysical tool for studying the cross-membrane translocation of ultralarge molecules and open new opportunities for analytical chemistry, synthetic biology, and nanomedicine.

Tetrahedral Framework Nucleic Acids: A Novel Strategy for Antibiotic Treating Drug-Resistant Infections.

Antibiotic multiresistance (AMR) has emerged as a major threat to human health as millions of people die from AMR-related problems every year. As has been witnessed during the global COVID-19 pandemic, the significantly increased demand for antibiotics has aggravated the issue of AMR. Therefore, there is an urgent need to find ways to alleviate it. Tetrahedral framework nucleic acids (tFNAs) are novel nanomaterials that are often used as drug delivery platforms because of their structural diversity. This study formed a tFNAs-antibiotic compound (TAC) which has a strong growth inhibitory effect on Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) in vitro owing to the increased absorption of antibiotics by bacteria and improved drug movement across cell membranes. We established a mouse model of systemic peritonitis and local wound infections. The TAC exhibited good biosafety and improved the survival rate of severely infected mice, promoting the healing of local infections. In addition to the better transport of antibiotics to the target, the TAC may also enhance immunity by regulating the differentiation of M1 and M2 macrophages, providing a new option for the treatment of infections.

The CNN model aided the study of the clinical value hidden in the implant images.

PURPOSE: This article aims to construct a new method to evaluate radiographic image identification results based on artificial intelligence, which can complement the limited vision of researchers when studying the effect of various factors on clinical implantation outcomes. METHODS: We constructed a convolutional neural network (CNN) model using the clinical implant radiographic images. Moreover, we used gradient-weighted class activation mapping (Grad-CAM) to obtain thermal maps to present identification differences before performing statistical analyses. Subsequently, to verify whether these differences presented by the Grad-CAM algorithm would be of value to clinical practices, we measured the bone thickness around the identified sites. Finally, we analyzed the influence of the implant type on the implantation according to the measurement results. RESULTS: The thermal maps showed that the sites with significant differences between Straumann BL and Bicon implants as identified by the CNN model were mainly the thread and neck area. (2) The heights of the mesial, distal, buccal, and lingual bone of the Bicon implant post-op were greater than those of Straumann BL (P < 0.05). (3) Between the first and second stages of surgery, the amount of bone thickness variation at the buccal and lingual sides of the Bicon implant platform was greater than that of the Straumann BL implant (P < 0.05). CONCLUSION: According to the results of this study, we found that the identified-neck-area of the Bicon implant was placed deeper than the Straumann BL implant, and there was more bone resorption on the buccal and lingual sides at the Bicon implant platform between the first and second stages of surgery. In summary, this study proves that using the CNN classification model can identify differences that complement our limited vision.

DNA-Origami NanoTrap for Studying the Selective Barriers Formed by Phenylalanine-Glycine-Rich Nucleoporins.

DNA nanotechnology provides a versatile and powerful tool to dissect the structure-function relationship of biomolecular machines like the nuclear pore complex (NPC), an enormous protein assembly that controls molecular traffic between the nucleus and cytoplasm. To understand how the intrinsically disordered, Phe-Gly-rich nucleoporins (FG-nups) within the NPC establish a selective barrier to macromolecules, we built a DNA-origami NanoTrap. The NanoTrap comprises precisely arranged FG-nups in an NPC-like channel, which sits on a baseplate that captures macromolecules that pass through the FG network. Using this biomimetic construct, we determined that the FG-motif type, grafting density, and spatial arrangement are critical determinants of an effective diffusion barrier. Further, we observed that diffusion barriers formed with cohesive FG interactions dominate in mixed-FG-nup scenarios. Finally, we demonstrated that the nuclear transport receptor, Ntf2, can selectively transport model cargo through NanoTraps composed of FxFG but not GLFG Nups. Our NanoTrap thus recapitulates the NPC's fundamental biological activities, providing a valuable tool for studying nuclear transport.

Bioswitchable Delivery of microRNA by Framework Nucleic Acids: Application to Bone Regeneration.

MicroRNAs (miRs) play an important role in regulating gene expression. Limited by their instabilities, miR therapeutics require delivery vehicles. Tetrahedral framework nucleic acids (tFNAs) are potentially applicable to drug delivery because they prominently penetrate tissue and are taken up by cells. However, tFNA-based miR delivery strategies have failed to separate the miRs after they enter cells, affecting miR efficiency. In this study, an RNase H-responsive sequence is applied to connect a sticky-end tFNA (stFNA) and miR-2861, which is a model miR, to target the expression of histone deacetylase 5 (HDAC5) in bone marrow mesenchymal stem cells. The resultant bioswitchable nanocomposite (stFNA-miR) enables efficient miR-2861 unloading and deployment after intracellular delivery, thereby inhibiting the expression of HDAC5 and promoting osteogenic differentiation. stFNA-miR also facilitated ideal bone repair via topical injection. In conclusion, a versatile miR delivery strategy is offered for various biomedical applications that necessitate modulation of gene expression.

Targeted and effective glioblastoma therapy via aptamer-modified tetrahedral framework nucleic acid-paclitaxel nanoconjugates that can pass the blood brain barrier.

Targeted DNA nanoparticles have been identified as one of the most promising nanocarriers in anti-glioma drug delivery. We established a multifunctional nanosystem for targeted glioma therapy. Tetrahedral framework nucleic acid (tFNA), entering U87MG cells and bEnd.3 cells, was chosen to deliver two aptamers, GMT8 and Gint4.T, and paclitaxel. GMT8 and Gint4.T, which specifically bind with U87MG cells and with PDGFRß, were linked with tFNA, to form Gint4.T-tFNA-GMT8 (GTG). GTG was efficiently internalized by U87MG and bEnd.3 cells and penetrated an in-vitro blood-brain-barrier model. GTG loaded with paclitaxel (GPC) had potentiated anti-glioma efficacy. It inhibited the proliferation, migration, and invasion of U87MG cells, and enhanced apoptosis induction in these cells. The expression of apoptosis-related proteins was significantly changed after treatment with GPC, confirming apoptosis induction. Our study demonstrated that the combination of GTG and paclitaxel has great potential for glioma treatment and tFNA shows great promise for use in drug delivery.

Hypoxia triggers angiogenesis by increasing expression of LOX genes in 3-D culture of ASCs and ECs.

OBJECTIVES: This study aimed to investigate the expression changes of LOX (lysyl oxidase) family genes, VEGFA, and VEGFB under hypoxic conditions in a co-culture system of ASCs (adipose-derived stromal cells) and ECs (endothelial cells). MATERIALS AND METHODS: ASCs and ECs were co-cultured under hypoxic and normal oxygen conditions in a 1:1 ratio, and the formation of vessel-like was detected at 7 days. The transwell co-culture system was used and cell lysates were collected at 7 days after co-culture in hypoxic and normal oxygen condition. Semi-quantitative PCR was performed to quantify the mRNA expression of VEGFA, VEGFB, and the LOX genes (LOX, LOXL-1, LOXL-2, LOXL-3, and LOXL-4). Expression changes were determined by densitomery. RESULTS: Enhanced angiogenesis was detected in the co-culture of ASCs and ECs under hypoxic conditions. Among the genes screened, VEGFA, VEGFB, LOXL-1, and LOXL-3 in ECs, both mono-cultured and co-cultured, were significantly enhanced after culturing under hypoxic conditions. In ASCs, VEGFB, LOXL-1, and LOXL-3 were upregulated. CONCLUSIONS: Contact co-culture between ASCs and ECs promotes angiogenesis under hypoxia. LOXL-1 and LOXL-3 expressions were increased in both ASCs and ECs and might play important roles in the enhanced angiogenesis promoted by hypoxia.

Effect of tetrahedral DNA nanostructures on proliferation and osteo/odontogenic differentiation of dental pulp stem cells via activation of the notch signaling pathway.

Dental pulp stem cells (DPSCs) derived from the human dental pulp tissue have multiple differentiation capabilities, such as osteo/odontogenic differentiation. Therefore, DPSCs are deemed as ideal stem cell sources for tissue regeneration. As new nanomaterials based on DNA, tetrahedral DNA nanostructures (TDNs) have tremendous potential for biomedical applications. Here, the authors aimed to explore the part played by TDNs in proliferation and osteo/odontogenic differentiation of DPSCs, and attempted to investigate if these cellular responses could be driven by activating the canonical Notch signaling pathway. Upon exposure to TDNs, proliferation and osteo/odontogenic differentiation of DPSCs were dramatically enhanced, accompanied by up regulation of Notch signaling. In general, our study suggested that TDNs can significantly promote proliferation and osteo/odontogenic differentiation of DPSCs, and this remarkable discovery can be applied in tissue engineering and regenerative medicine to develop a significant and novel method for bone and dental tissue regeneration.

Reconstruction of Mandible: A Fully Digital Workflow From Visualized Iliac Bone Grafting to Implant Restoration.

PURPOSE: Although digital aids can help surgeons compensate for the shortcomings of traditional mandibular reconstruction techniques to perform surgery more precisely and effectively, the use of these digital techniques has often been fragmented, divided, and incomplete. This article describes the workflow of a fully digital mandibular reconstruction to explore the proper indications and discusses innovations based on the accuracy and effectiveness of digital techniques. MATERIALS AND METHODS: A restoration-oriented mandibular reconstruction was performed by applying different digital techniques. Preoperative virtual surgery and rapid prototyping were used to aid the vascularized iliac bone graft surgery, which offered a solid basis for the ensuing treatment. Subsequently, implant rehabilitation was accomplished with the assistance of computer-assisted design and manufacture, laser treatment, and selective laser melting techniques. RESULT: The workflow of the fully digital mandibular reconstruction successfully achieved a restoration-oriented treatment. These predictable, accurate, and effective digital techniques improved the consistency of pretreatment design and follow-up treatment. The treatment sequence achieved high predictability and reproducibility owing to the use of digital techniques. CONCLUSION: This study shows that a digital workflow can be predictable, accurate, and effective, which suggests that it could be a valid digital protocol for developing a treatment sequence for patients with jaw defects caused by trauma, congenital anomalies, or mandibular tumor resection.

A Novel Bioswitchable miRNA Mimic Delivery System: Therapeutic Strategies Upgraded from Tetrahedral Framework Nucleic Acid System for Fibrotic Disease Treatment and Pyroptosis Pathway Inhibition.

There has been considerable interest in gene vectors and their role in regulating cellular activities and treating diseases since the advent of nucleic acid drugs. MicroRNA (miR) therapeutic strategies are research hotspots as they regulate gene expression post-transcriptionally and treat a range of diseases. An original tetrahedral framework nucleic acid (tFNA) analog, a bioswitchable miR inhibitor delivery system (BiRDS) carrying miR inhibitors, is previously established; however, it remains unknown whether BiRDS can be equipped with miR mimics. Taking advantage of the transport capacity of tetrahedral framework nucleic acid (tFNA) and upgrading it further, the treatment outcomes of a traditional tFNA and BiRDS at different concentrations on TGF-ß- and bleomycin-induced fibrosis simultaneously in vitro and in vivo are compared. An upgraded traditional tFNA is designed by successfully synthesizing a novel BiRDS, carrying a miR mimic, miR-27a, for treating skin fibrosis and inhibiting the pyroptosis pathway, which exhibits stability and biocompatibility. BiRDS has three times higher efficiency in delivering miRNAs than the conventional tFNA with sticky ends. Moreover, BiRDS is more potent against fibrosis and pyroptosis-related diseases than tFNAs. These findings indicate that the BiRDS can be applied as a drug delivery system for disease treatment.

DNA framework signal amplification platform-based high-throughput systemic immune monitoring.

Systemic immune monitoring is a crucial clinical tool for disease early diagnosis, prognosis and treatment planning by quantitative analysis of immune cells. However, conventional immune monitoring using flow cytometry faces huge challenges in large-scale sample testing, especially in mass health screenings, because of time-consuming, technical-sensitive and high-cost features. However, the lack of high-performance detection platforms hinders the development of high-throughput immune monitoring technology. To address this bottleneck, we constructed a generally applicable DNA framework signal amplification platform (DSAP) based on post-systematic evolution of ligands by exponential enrichment and DNA tetrahedral framework-structured probe design to achieve high-sensitive detection for diverse immune cells, including CD4+, CD8+ T-lymphocytes, and monocytes (down to 1/100 µl). Based on this advanced detection platform, we present a novel high-throughput immune-cell phenotyping system, DSAP, achieving 30-min one-step immune-cell phenotyping without cell washing and subset analysis and showing comparable accuracy with flow cytometry while significantly reducing detection time and cost. As a proof-of-concept, DSAP demonstrates excellent diagnostic accuracy in immunodeficiency staging for 107 HIV patients (AUC > 0.97) within 30 min, which can be applied in HIV infection monitoring and screening. Therefore, we initially introduced promising DSAP to achieve high-throughput immune monitoring and open robust routes for point-of-care device development.

A Bioswitchable MiRNA Delivery System: Tetrahedral Framework DNA-Based miRNA Delivery System for Applications in Wound Healing.

The human body's primary line of defense, the skin, is especially prone to harm. Although microRNA (miRNA)-based therapies have attracted increasing attention for skin wound healing, their applications remain limited owing to a range of issues. Tetrahedral framework DNA (tFNA), a nanomaterial possessing nucleic acid characteristics, exhibits an excellent biocompatibility, in addition to anti-inflammatory and transdermal delivery capabilities, and can accelerate skin wound healing. Due to its potential to exert synergistic action with therapeutic miRNA, tFNA has been considered an ideal vehicle for miRNA therapy. The design and synthesis of a bioswitchable miRNA delivery system (BiRDS) is reported, which contains three miRNAs as well as a nucleic acid core to maximize the loading capacity while preserving the characteristics of tFNA. A high stability, excellent permeability of cells as well as tissues and good biological compatibility are demonstrated. By selectively inhibiting heparin-binding epidermal growth factor (HB-EGF), the BiRDS can inhibit the NF-κB pathway while simultaneously controlling the PTEN/Akt pathway. As a result, the BiRDS helps wound healing go through the inflammation to the proliferative phase. This study demonstrates the advantages of the BiRDS in miRNA-based therapy and provides new research ideas for the treatment of skin-related diseases.

A minimally invasive method for titanium mesh fixation with resorbable sutures in guided bone regeneration: A retrospective study.

OBJECTIVES: Titanium mesh has become a mainstream choice for guided bone regeneration (GBR) owing to its excellent space maintenance. However, the traditional fixation method using titanium screws impacts surgery efficiency and increases patient trauma. We report a novel method of fixing a titanium mesh using resorbable sutures. We assessed the feasibility of resorbable sutures for fixing a titanium mesh and whether it can serve as a stable, universal, and minimally invasive fixation method for a broader application of titanium meshes. METHODS: Patients undergoing GBR with a digital titanium mesh fixed using titanium screws (TS group) and resorbable sutures (RS group) were observed at different time points. The stability of the fixation methods was evaluated on parameters such as titanium mesh spatial displacement, bone augmentation, and bone resorption. RESULTS: A total of 36 patients were included in this study. The exposure rate of the titanium mesh in the TS group was 16.67%, while no exposure was noted in the RS group. There was no significant difference in the parameters of titanium mesh spatial displacement, bone augmentation, and bone resorption between the two groups (p > 0.05). CONCLUSION: The use of resorbable sutures for fixing a titanium mesh can achieve similar results to traditional fixation using titanium screws. Although this new fixation method can improve the efficiency of the surgery and reduce the risk of complications, the long-term clinical effects require further follow-up investigation.

A dynamic DNA tetrahedron framework for active targeting.

An active targeting strategy-enabled DNA tetrahedron delivery vehicle could facilitate stable drug encapsulation and stimuli-responsive on-demand release, building a universal platform for different drug delivery requirements. Owing to the excellent biocompatible nature, programmability and remarkable cell and tissue permeability, the tetrahedral DNA nanostructure (TDN) has proven its value in the delivery of various bioactive molecules. We previously described this as a static multifunctional complex in our earlier protocol. However, static structures and passive targeting behavior might introduce off-target effects under complicated biological conditions. Therefore, in this Protocol Extension, we present a major update of the TDN delivery vehicle enabling an active targeting strategy to be used for stimuli-sensitive conformation changes and on-site cargo release, which could avoid drawbacks, including complex and time-consuming fabrication processes and undetermined cell penetration ability of other DNA-based delivery vehicles. Upon exquisite design of TDN size based on cargo type, one-pot annealing is applied to fabricate the Tiamat-designed TDN exoskeleton. Then the design of the dynamic DNA apparatus can be based on the target and environmental stimuli, including DNA strand hybridization-based and pH-sensitive DNA apparatus, and careful titration of strand lengths and mismatches is achieved using polyacrylamide and agarose gel electrophoresis, or fluorophore modifications. Finally, cargo loading strategies are designed, including site and stand titration and cargo encapsulation verification. The dynamic structures show promising targetability and effectiveness in antitumor and anti-inflammatory treatment in vitro and in vivo. Assembly and characterization in the lab takes ~5 d, and the timing for the verification of biostability and biological applications depends on the uses.

Tetrahedral framework nucleic acid loaded with glabridin: A transdermal delivery system applicated to anti-hyperpigmentation.

Topical application of tyrosinase inhibitors, such as hydroquinone and arbutin, is the most common clinical treatment for hyperpigmentation. Glabridin (Gla) is a natural isoflavone that inhibits tyrosinase activity, free radical scavenging, and antioxidation. However, its water solubility is poor, and it cannot pass through the human skin barrier alone. Tetrahedral framework nucleic acid (tFNA), a new type of DNA biomaterial, can penetrate cells and tissues and can be used as carriers to deliver small-molecule drugs, polypeptides, and oligonucleotides. This study aimed to develop a compound drug system using tFNA as the carrier to transport Gla and deliver it through the skin to treat pigmentation. Furthermore, we aimed to explore whether tFNA-Gla can effectively alleviate the hyperpigmentation caused by increased melanin production and determine whether tFNA-Gla exerts substantial synergistic effects during treatment. Our results showed that the developed system successfully treated pigmentation by inhibiting regulatory proteins related to melanin production. Furthermore, our findings showed that the system was effective in treating epidermal and superficial dermal diseases. The tFNA-based transdermal drug delivery system can thus develop into novel, effective options for non-invasive drug delivery through the skin barrier.

Liver-Targeted Delivery of Small Interfering RNA of C-C Chemokine Receptor 2 with Tetrahedral Framework Nucleic Acid Attenuates Liver Cirrhosis.

Liver cirrhosis is the end stage of chronic liver diseases without approved clinical drugs. In this study, a new strategy that uses a C-C chemokine receptor 2 (CCR2) small interfering RNA silencing (siCcr2)-based therapy by loading multivalent siCcr2 with tetrahedron framework DNA nanostructure (tFNA) vehicle (tFNA-siCcr2) was established to attenuate liver fibrosis. tFNA-siCcr2 was successfully synthesized without changing the physiochemical properties of tFNA. Compared to the naked siCcr2 molecule, the tFNA-siCcr2 complex altered the accumulation from the kidney to the liver after the intraperitoneal injection. The tFNA-siCcr2 complex also prolonged hepatic retention and mainly colocalized within macrophages and endothelial cells. tFNA-siCcr2 efficiently silenced CCR2 and significantly ameliorated liver fibrosis in prevention and treatment interventions. Single-cell RNA sequencing followed by experimental validation suggested that tFNA-siCcr2 can restore the immune cell landscape and construct an antifibrotic niche by inhibiting profibrotic macrophage and neutrophil accumulation in the murine fibrotic liver. Molecularly, the tFNA-siCcr2 complex reduced inflammatory mediator production by inactivating the NF-κB signaling pathway. In conclusion, the tFNA-based liver-targeted tFNA-siCcr2 delivery complex efficiently ameliorated liver fibrosis by restoring the immune cell landscape and constructing an antifibrotic niche, which makes the tFNA-siCcr2 complex a potential therapeutic candidate for the clinical treatment of liver cirrhosis.

Modeling HIV-1 nuclear entry with nucleoporin-gated DNA-origami channels.

Delivering the virus genome into the host nucleus through the nuclear pore complex (NPC) is pivotal in human immunodeficiency virus 1 (HIV-1) infection. The mechanism of this process remains mysterious owing to the NPC complexity and the labyrinth of molecular interactions involved. Here we built a suite of NPC mimics-DNA-origami-corralled nucleoporins with programmable arrangements-to model HIV-1 nuclear entry. Using this system, we determined that multiple cytoplasm-facing Nup358 molecules provide avid binding for capsid docking to the NPC. The nucleoplasm-facing Nup153 preferentially attaches to high-curvature regions of the capsid, positioning it for tip-leading NPC insertion. Differential capsid binding strengths of Nup358 and Nup153 constitute an affinity gradient that drives capsid penetration. Nup62 in the NPC central channel forms a barrier that viruses must overcome during nuclear import. Our study thus provides a wealth of mechanistic insight and a transformative toolset for elucidating how viruses like HIV-1 enter the nucleus.

Prospects and challenges of dynamic DNA nanostructures in biomedical applications.

The physicochemical nature of DNA allows the assembly of highly predictable structures via several fabrication strategies, which have been applied to make breakthroughs in various fields. Moreover, DNA nanostructures are regarded as materials with excellent editability and biocompatibility for biomedical applications. The ongoing maintenance and release of new DNA structure design tools ease the work and make large and arbitrary DNA structures feasible for different applications. However, the nature of DNA nanostructures endows them with several stimulus-responsive mechanisms capable of responding to biomolecules, such as nucleic acids and proteins, as well as biophysical environmental parameters, such as temperature and pH. Via these mechanisms, stimulus-responsive dynamic DNA nanostructures have been applied in several biomedical settings, including basic research, active drug delivery, biosensor development, and tissue engineering. These applications have shown the versatility of dynamic DNA nanostructures, with unignorable merits that exceed those of their traditional counterparts, such as polymers and metal particles. However, there are stability, yield, exogenous DNA, and ethical considerations regarding their clinical translation. In this review, we first introduce the recent efforts and discoveries in DNA nanotechnology, highlighting the uses of dynamic DNA nanostructures in biomedical applications. Then, several dynamic DNA nanostructures are presented, and their typical biomedical applications, including their use as DNA aptamers, ion concentration/pH-sensitive DNA molecules, DNA nanostructures capable of strand displacement reactions, and protein-based dynamic DNA nanostructures, are discussed. Finally, the challenges regarding the biomedical applications of dynamic DNA nanostructures are discussed.

Functionalizing Framework Nucleic-Acid-Based Nanostructures for Biomedical Application.

Strategies for functionalizing diverse tetrahedral framework nucleic acids (tFNAs) have been extensively explored since the first successful fabrication of tFNA by Turberfield. One-pot annealing of at least four DNA single strands is the most common method to prepare tFNA, as it optimizes the cost, yield, and speed of assembly. Herein, the focus is on four key merits of tFNAs and their potential for biomedical applications. The natural ability of tFNA to scavenge reactive oxygen species, along with remarkable enhancement in cellular endocytosis and tissue permeability based on its appropriate size and geometry, promotes cell-material interactions to direct or probe cell behavior, especially to treat inflammatory and degenerative diseases. Moreover, the structural programmability of tFNA enables the development of static tFNA-based nanomaterials via engineering of functional oligonucleotides or therapeutic molecules, and dynamic tFNAs via attachment of stimuli-responsive DNA apparatuses, leading to potential applications in targeted therapies, tissue regeneration, antitumor strategies, and antibacterial treatment. Although there are impressive performance and significant progress, the challenges and prospects of functionalizing tFNA-based nanostructures are still indicated in this review.