Immunosuppressive tumor microenvironment and limited intratumoral permeation have largely constrained the outcome of tumor therapy. Herein, we report a tailored DNA structure-based nanoplatform with striking tumor-penetrating capability for targeted remodeling of immunosuppressive tumor microenvironment in vivo. In our design, chemo-immunomodulator (gemcitabine) can be precisely grafted in DNA sequences via a reactive oxygen species (ROS)-sensitive linker. After self-assembly, the gemcitabine-grafted DNA structure can site-specifically organize legumain-activatable melittin pro-peptide (promelittin) on each vertex for intratumoral delivery and further function as the template to load photosensitizers (methylene blue) for ROS production. The tailored DNA nanoplatform can achieve targeted accumulation, highly improved intratumoral permeation, and efficient immunogenic cell death of tumor cells by laser irradiation. Finally, the immunosuppressive tumor microenvironment can be successfully remodeled by reducing multi-type immunosuppressive cells and enhancing the infiltration of cytotoxic lymphocytes in the tumor. This rationally developed multifunctional DNA nanoplatform provides a new avenue for the development of tumor therapy.

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Research on self-assembled deoxyribonucleic acid (DNA) nanostructures with different shapes, sizes, and functions has recently made rapid progress owing to its biocompatibility, programmability, and stability. Among these, triangular unit-based DNA nanostructures, which are typically multi-arm DNA tiles, have been widely applied because of their unique structural rigidity, spatial flexibility, and cell permeability. Triangular unit-based DNA nanostructures are folded from multiple single-stranded DNA using the principle of complementary base pairing. Its shape and size can be determined using pre-set scaffold strands, segmented base complementary regions, and sequence lengths. The resulting DNA nanostructures retain the desired sequence length to serve as binding sites for other molecules and obtain satisfactory results in molecular recognition, spatial orientation, and target acquisition. Therefore, extensive research on triangular unit-based DNA nanostructures has shown that they can be used as powerful tools in the biosensing field to improve specificity, sensitivity, and accuracy. Over the past few decades, various design strategies and assembly techniques have been established to improve the stability, complexity, functionality, and practical applications of triangular unit-based DNA nanostructures in biosensing. In this review, we introduce the structural design strategies and principles of typical triangular unit-based DNA nanostructures, including triangular, tetrahedral, star, and net-shaped DNA. We then summarize the functional properties of triangular unit-based DNA nanostructures and their applications in biosensing. Finally, we critically discuss the existing challenges and future trends.

Compared to conventional nucleic acid detection methods, label-free single nucleotide polymorphism (SNP) detection presents challenging due to the necessity of discerning single base mismatches, especially in the field of enzyme-free detection. In this study, we introduce a novel bulged-type DNA duplex probe designed to significantly amplify single-base differences. This probe is integrated with programmable DNA-based nanostructures to develop a sensitive, label-free biosensor for nonenzymatic SNP detection. The duplex probe with one bulge could selectively identify wild-typed DNA (WT) and mutant-type DNA (MT) based on a competitive strand displacement reaction mechanism. The hyperbranched HCR (HHCR) by incorporating of hairpin DNA into the DNA tetrahedron and surface-tethering on the portable screen printing electrode (SPCE) significantly favor the formation of negatively charged DNA nanostructure. We harnessed strong repulsion of DNA nanostructure towards the electroactive [Fe(CN)₆]³⁻/⁴⁻ in combination with electrochemical technique to create a label-free biosensor. This simple, enzyme-free and label-free biosensor could detect MT with a detection limit of 56 aM, even in multiple sequence backgrounds. The study served as the proof-of-concept for the integration of enzyme-free competitive mechanism and label-free strategy, which can be extended as a powerful tool to various fields.

DNA has been employed as building blocks for the construction of nanomaterials due to their programmability and wide range applications. The functional branched DNA (bDNA) nanostructure is largely dependent on the sequence and structural symmetry. Despite the discovery of different structures, the synthesis of bDNA nanostructures from optimal number of oligonucleotides is yet to be explored. In the current study, for the first time we demonstrate the designing of stable monomeric bDNA structures using two or three oligonucleotides. Furthermore, the stability of bDNA nanostructures was thoroughly investigated in presence of different pH, cations, fetal bovine serum and DNase I. The thermodynamic parameters indicated that hydrogen bonding and van der Waals interactions played a major role during self-assembly of bDNA nanostructures. From the gel retardation assay, we confirmed the binding of complementary oligonucleotides to the bDNA nanostructures, thus can be explored for target specific transcript regulation. In conclusion, the self-assembled DNA nanostructures developed from optimal oligonucleotides are stable in physiological environment and can be used for biomedical applications.

Recent studies have revealed that liquid-liquid phase separation (LLPS) plays crucial roles in various cellular functions. Droplets formed via LLPS within cells, often referred to as membraneless organelles, serve to concentrate specific molecules, thus enhancing biochemical reactions. Artificial LLPS systems have been utilized to construct synthetic cell models, employing a range of synthetic molecules. LLPS systems based on DNA nanotechnology are particularly notable for their designable characteristics in droplet formation, dynamics, properties, and functionalities. This review surveys recent advancements in DNA-based LLPS systems, underscoring the programmability afforded by DNA\'s base-pair specific interactions. We discuss the fundamentals of DNA droplet formation, including temperature-dependence and physical properties, along with the precise control achievable through sequence design. Attention is given to the phase separation of DNA nanostructures on two-dimensional closed interfaces, which results in spatial pattern formation at the interface. Furthermore, we spotlight the potential of DNA droplet computing for cancer diagnostics through specific microRNA pattern recognition. We envision that DNA-based LLPS presents a versatile platform for the exploration of cellular mimicry and opens innovative ways for the development of functional synthetic cells.

It was previously demonstrated that polypod-like nanostructured DNA (polypodna) comprising three or more oligodeoxynucleotides (ODNs) were useful for the delivery of ODNs containing cytosine-phosphate-guanine (CpG) motifs, or CpG ODNs, to immune cells. Although the immunostimulatory activity of single-stranded CpG ODNs is highly dependent on CpG motif sequence and position, little is known about how the position of the motif affects the immunostimulatory activity of CpG motif-containing nanostructured DNAs. In the present study, four series of polypodna were designed, each comprising a CpG ODN with one potent CpG motif at varying positions and 2-5 CpG-free ODNs, and investigated their immunostimulatory activity using Toll-like receptor-9 (TLR9)-positive murine macrophage-like RAW264.7 cells. Polypodnas with the CpG motif in the 5\'-overhang induced more tumor necrosis factor-α release than those with the motif in the double-stranded region, even though their cellular uptake were similar. Importantly, the rank order of the immunostimulatory activity of single-stranded CpG ODNs changed after their incorporation into polypodna. These results indicate that the CpG ODN sequence as well as the motif location in nanostructured DNAs should be considered for designing the CpG motif-containing nanostructured DNAs for immune stimulation.

Zeta potential is commonly referred as surface charge density and is a key factor in modulating the structural and functional properties of nucleic acids. Although the negative charge density of B-DNA is well understood, there is no prior description of the zeta potential measurement of Z-DNA. In this study, for the first time we discover the zeta potential difference between B-DNA and lanthanum chloride-induced Z-DNA. A series of linear repeat i.e. (CG)n and (GC)n DNA as well as branched DNA (bDNA) structures was used for the B-to-Z DNA transition. Herein, the positive zeta potential of Z-DNA has been demonstrated as a powerful tool to discriminate between B-form and Z-form of DNA. The generality of the approach has been validated both in linear and bDNA nanostructures. Thus, we suggest zeta potential can be used as an ideal signature for the left-handed Z-DNA.

DNA nanostructures (DNs) have found increasing use in biosensing, drug delivery, and therapeutics because of their customizable assembly, size and shape control, and facile functionalization. However, their limited cellular uptake and nuclear delivery have hindered their effectiveness in these applications. Here, we demonstrate the potential of applying cell-surface binding as a general strategy to enable rapid enhancement of intracellular and intranuclear delivery of DNs. By targeting the plasma membrane via cholesterol anchors or the cell-surface glycocalyx using click chemistry, we observe a significant 2 to 8-fold increase in the cellular uptake of three distinct types of DNs that include nanospheres, nanorods, and nanotiles, within a short time frame of half an hour. Several factors are found to play a critical role in modulating the uptake of DNs, including their geometries, the valency, positioning and spacing of binding moieties. Briefly, nanospheres are universally preferable for cell surface attachment and internalization. However, edge-decorated nanotiles compensate for their geometry deficiency and outperform nanospheres in both categories. In addition, we confirm the short-term structural stability of DNs by incubating them with cell medium and cell lysate. Further, we investigate the endocytic pathway of cell-surface bound DNs and reveal that it is an interdependent process involving multiple pathways, similar to those of unmodified DNs. Finally, we demonstrate that cell-surface attached DNs exhibit a substantial enhancement in the intranuclear delivery. Our findings present an application that leverages cell-surface binding to potentially overcome the limitations of low cellular uptake, which may strengthen and expand the toolbox for effective cellular and nuclear delivery of DNA nanostructure systems.

Developing sensitive and accurate Ochratoxin A (OTA) detection methods is essential for food safety. Herein, a simple and reliable strategy for regulating interenzyme distance based on a rigid DNA quadrangular prism as a scaffold was proposed to establish a new electrochemical biosensor for ultrasensitive detection of OTA. The interenzyme distances were precisely adjusted by changing the sequences of the hybridized portions of hairpins SH1 and SH2 to the DNA quadrangular prism, avoiding the complexity and instability of the previous DNA scaffold-based enzyme spacing adjustment strategies. The electrochemical biosensor constructed at the optimal interenzyme distance (10.4 nm) achieved sensitive detection of OTA in a dynamic concentration range from 10 fg/mL to 250 ng/mL with a detection limit of 3.1 fg/mL. In addition, the biosensor was applied to quantify OTA in real samples, exhibiting great application potential in food safety.