Monodispersed and Monofunctionalized DNA-Caged Au Nano-Clusters with Enhanced Optical Properties for STED Imaging.

The performance of Stimulated Emission Depletion (STED) microscopy depends critically on the fluorescent probe. Ultrasmall Au nanoclusters (Au NCs) exhibit large Stokes shift, and good stimulated emission response, which are potentially useful for STED imaging. However, Au NCs are polydispersed in size, sensitive to the surrounding environment, and difficult to control surface functional group stoichiometry, which results in reduced density and high heterogeneity in the labeling of biological structures. Here, this limitation is overcome by developing a method to encapsulate ultrasmall Au NCs with DNA cages, which yielded monodispersed, and monofunctionalized Au NCs that are long-term stable. Moreover, the DNA-caging also greatly improved the fluorescence quantum yield and photostability of Au NCs. In STED imaging, the DNA-caged Au NCs yielded ≈40 nm spatial resolution and are able to resolve microtubule line shapes with good labeling density and homogeneity. In contrast, without caging, the Au NCs-DNA conjugates only achieved ≈55 nm resolution and yielded spotted, poorly resolved microtubule structures, due to the presence of aggregates. Overall, a method is developed to achieve precise surface functionalization and greatly improve the monodispersity, stability, as well as optical properties of Au NCs, providing a promising class of fluorescent probes for STED imaging.

Cholesterol-Modified DNA Nanostructures Serve as Effective Non-Viral Carriers for Delivering siRNA to the Kidneys to Prevent Acute Kidney Injury.

With the emergence of gene therapy utilizing viral vectors, the potential risks associated with these vectors have prompted increased attention toward non-viral alternatives. DNA nanotechnology enables the assembly of specific oligonucleotide chains into nanostructures possessing defined spatial configurations. Due to their inherent characteristics, DNA nanostructures possess natural advantages as carriers for regulating gene expression in a non-viral manner. Cholesterol modification can convert DNA nanostructures from hydrophilic materials to amphiphilic materials, thereby extending their systemic circulation time. In this study, the high-dimensional design and cholesterol modification are shown to prolong the systemic circulation half-life of DNA nanostructures in mice. Specifically, the tetrahedron structure modified with three cholesterol molecules (TDN-3Chol) exhibit excellent circulation time and demonstrate a preference for renal uptake. The unique characteristics of TDN-3Chol can effectively deliver p53 siRNA to the mouse renal tubular tissue, resulting in successful knockdown of p53 and demonstrating its potential for preventing acute kidney injury. Furthermore, TDN-3Chol is not exhibited significant toxicity in mice, highlighting its promising role as a non-viral vector for targeted gene expression regulation in the kidneys. The designed non-viral vector as a prophylactic medication shows potential in addressing the current clinical challenges associated with nephrotoxic drugs.

Securing LYTAC with Logic-Identification System for Cancer Cell-Selective Membrane Protein Degradation.

Lysosome-targeting chimera (LYTAC) links proteins of interest (POIs) with lysosome-targeting receptors (LTRs) to achieve membrane protein degradation, which is becoming a promising therapeutic modality. However, cancer cell-selective membrane protein degradation remains a big challenge considering expressions of POIs in both cancer cells and normal cells, as well as broad tissue distribution of LTRs. Here a logic-identification system is designed, termed Logic-TAC, based on cell membrane-guided DNA calculations to secure LYTAC selectively for cancer cells. Logic-TAC is designed as a duplex DNA structure, with both POI and LTR recognition regions sealed to avoid systematic toxicity during administration. MCF-7 and MCF-10A are chosen as sample cancer cell and normal cell respectively. As input 1 for logic-identification, membrane proteins EpCAM, which is highly expressed by MCF-7 but barely by MCF-10A, reacts with Logic-TAC to expose POI recognition region. As input 2 for logic-identification, Logic-TAC binds to POI, membrane protein MUC1, to expose LTR recognition region. As output, MUC1 is connected to LTR and degraded via lysosome pathway selectively for cancer cell MCF-7 with little side effect on normal cell MCF-10A. The logic-identification system also demonstrated satisfactory in vivo therapeutic results, indicating its promising potential in precise targeted therapy.

Myeloid cell-specific topoisomerase 1 inhibition using DNA origami mitigates neuroinflammation.

Targeting myeloid cells, especially microglia, for the treatment of neuroinflammatory diseases such as multiple sclerosis (MS), is underappreciated. Our in silico drug screening reveals topoisomerase 1 (TOP1) inhibitors as promising drug candidates for microglial modulation. We show that TOP1 is highly expressed in neuroinflammatory conditions, and TOP1 inhibition using camptothecin (CPT) and its FDA-approved analog topotecan (TPT) reduces inflammatory responses in microglia/macrophages and ameliorates neuroinflammation in vivo. Transcriptomic analyses of sorted microglia from LPS-challenged mice reveal an altered transcriptional phenotype following TPT treatment. To target myeloid cells, we design a nanosystem using ß-glucan-coated DNA origami (MyloGami) loaded with TPT (TopoGami). MyloGami shows enhanced specificity to myeloid cells while preventing the degradation of the DNA origami scaffold. Myeloid-specific TOP1 inhibition using TopoGami significantly suppresses the inflammatory response in microglia and mitigates MS-like disease progression. Our findings suggest that TOP1 inhibition in myeloid cells represents a therapeutic strategy for neuroinflammatory diseases and that the myeloid-specific nanosystems we designed may also benefit the treatment of other diseases with dysfunctional myeloid cells.

Bimodal DNA self-origami material with nucleic acid function enhancement.

BACKGROUND: The design of DNA materials with specific nanostructures for biomedical tissue engineering applications remains a challenge. High-dimensional DNA nanomaterials are difficult to prepare and are unstable; moreover, their synthesis relies on heavy metal ions. Herein, we developed a bimodal DNA self-origami material with good biocompatibility and differing functions using a simple synthesis method. We simulated and characterized this material using a combination of oxDNA, freeze-fracture electron microscopy, and atomic force microscopy. Subsequently, we optimized the synthesis procedure to fix the morphology of this material. RESULTS: Using molecular dynamics simulation, we found that the bimodal DNA self-origami material exhibited properties of spontaneous stretching and curling and could be fixed in a single morphology via synthesis control. The application of different functional nucleic acids enabled the achievement of various biological functions, and the performance of functional nucleic acids was significantly enhanced in the material. Consequently, leveraging the various functional nucleic acids enhanced by this material will facilitate the attainment of diverse biological functions. CONCLUSION: The developed design can comprehensively reveal the morphology and dynamics of DNA materials. We thus report a novel strategy for the construction of high-dimensional DNA materials and the application of functional nucleic acid-enhancing materials.

An impedance labeling free electrochemical aptamer sensor based on tetrahedral DNA nanostructures for doxorubicin determination.

Based on the electrochemical impedance method, a marker-free biosensor with aptamer as a biometric element was developed for the determination of doxorubicin (DOX). By combining aptamer with rigid tetrahedral DNA nanostructures (TDNs) and fixing them on the surface of gold electrode (GE) as biometric elements, the density and directivity of surface nanoprobes improved, and DOX was captured with high sensitivity and specificity. DOX was captured by immobilized aptamers on the GE, which inhibited electron transfer between the GE and [Fe(CN)6]3-/4- in solution, resulting in a change in electrochemical impedance. When the DOX concentration was between 10.0 and 100.0 nM, the aptasensor showed a linear relationship with charge transfer resistance, the relative standard deviation (RSD) ranged from 3.6 to 5.9%, and the detection limit (LOD) was 3.0 nM. This technique offered a successful performance for the determination of the target analyte in serum samples with recovery in the range 97.0 to 99.6% and RSD ranged from 4.8 to 6.5%. This method displayed the advantages of fast response speed, good selectivity, and simple sensor structure and showed potential application in therapeutic drug monitoring.

Reversible Regulation of Long-Distance Charge Transport in DNA Nanowires by Dynamically Controlling Steric Conformation.

Understanding of DNA-mediated charge transport (CT) is significant for exploring circuits at the molecular scale. However, the fabrication of robust DNA wires remains challenging due to the persistence length and natural flexibility of DNA molecules. Moreover, CT regulation in DNA wires often relies on predesigned sequences, which limit their application and scalability. Here, we addressed these issues by preparing self-assembled DNA nanowires with lengths of 30-120 nm using structural DNA nanotechnology. We employed these nanowires to plug individual gold nanoparticles into a circuit and measured the transport current in nanowires with an optical imaging technique. Contrary to the reported cases with shallow or no length dependence, a fair current attenuation was observed with increasing nanowire length, which experimentally confirmed the prediction of the incoherent hopping model. We also reported a mechanism for the reversible CT regulation in DNA nanowires, which involves dynamic transitions in the steric conformation.

DNA Nanostructures: Self-Adjuvant Carriers for Highly Efficient Subunit Vaccines.

Subunit vaccines based on antigen proteins or epitopes of pathogens or tumors show advantages in immunological precision and high safety, but are often limited by their low immunogenicity. Adjuvants can boost immune responses by stimulating immune cells or promoting antigen uptake by antigen presenting cells (APCs), yet existing clinical adjuvants struggle in simultaneously achieving these dual functions. Additionally, the spatial organization of antigens might be crucial to their immunogenicity. Hence, superior adjuvants should potently stimulate the immune system, precisely arrange antigens, and effectively deliver antigens to APCs. Recently, precisely organizing and delivering antigens with the unique editability of DNA nanostructures has been proposed, presenting unique abilities in significantly improving the immunogenicity of antigens. In this minireview, we will discuss the principles behind using DNA nanostructures as self-adjuvant carriers and review the latest advancements in this field. The potential and challenges associated with self-adjuvant DNA nanostructures will also be discussed.

DNA Framework-Templated Fabrication of Ultrathin Electroactive Gold Nanosheets.

Generally, two-dimensional gold nanomaterials have unique properties and functions that offer exciting application prospects. However, the crystal phases of these materials tend to be limited to the thermodynamically stable crystal structure. Herein, we report a DNA framework-templated approach for the ambient aqueous synthesis of freestanding and microscale amorphous gold nanosheets with ultrathin sub-nanometer thickness. We observe that extended single-stranded DNA on DNA nanosheets can induce site-specific metallization and enable precise modification of the metalized nanostructures at predefined positions. More importantly, the as-prepared gold nanosheets can serve as an electrocatalyst for glucose oxidase-catalyzed aerobic oxidation, exhibiting enhanced electrocatalytic activity (~3-fold) relative to discrete gold nanoclusters owing to a larger electrochemical active area and wider band gap. The proposed DNA framework-templated metallization strategy is expected to be applicable in a broad range of fields, from catalysis to new energy materials.

A Highly Tumor Permeating DNA Nanoplatform for Efficient Remodeling of Immunosuppressive Tumor Microenvironments.

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.

Surfactant-Assisted Purification of Hydrophobic DNA Nanostructures.

Purification of functional DNA nanostructures is an essential step in achieving intended functions because misfolded structures and the remaining free DNA strands in a solution can interact and affect their behavior. However, due to hydrophobicity-mediated aggregation, it is difficult to purify DNA nanostructures modified with hydrophobic molecules by conventional methods. Herein, we report the purification of cholesterol-modified DNA nanostructures by using a novel surfactant-assisted gel extraction. The addition of sodium cholate (SC) to the sample solution before structure folding prevented aggregation; this was confirmed by gel electrophoresis. We also found that adding sodium dodecyl sulfate (SDS) to the sample inhibited structural folding. The cholesterol-modified DNA nanostructures prepared with SC were successfully purified by gel extraction, and their ability to bind to the lipid membrane surfaces was maintained. This method will facilitate the purification of DNA nanostructures modified with hydrophobic molecules and expand their applicability in the construction of artificial cell-like systems.

A Novel Fluorescent Aptasensor Based on Dual-labeled DNA Nanostructure for Simultaneous Detection of Ochratoxin A and Aflatoxin B1.

Based on DNA strand replacement reaction and aptamer-specific recognition, a simple dual-labeled DNA nanostructure is designed for the simultaneous detection of Ochratoxin A (OTA) and aflatoxin B1 (AFB1). C1 is labeled with Cy3 and Cy5, while C2 and C3 are labeled with BHQ2. The fluorescence intensity of DNA nanostructure composed of C1, C2 and C3 is weak because of fluorescence resonance energy transfer. When OTA Aptamer (OTA-Apt) and AFB1 Aptamer (AFB1-Apt) are added to the homogeneous system at the same time, C1 can be replaced with the help of toehold strand displacement, resulting in fluorescence enhancement. In the presence of both OTA and AFB1, the toehold strand displacement reaction is inhibited due to preferential binding between the target and their corresponding aptamers. The limit of detection of OTA was 0.007 ng/mL and that of AFB1 was 0.03 ng/mL. The recoveries of OTA and AFB1 were 96%-101% and 97%-101% in the corn sample, and 99%-101% and 92%-106% in the wine sample. Compared with other sensors, the preparation of this aptasensor needs simpler experimental steps and a shorter total-preparing time, confirming the convenient, rapid, and time-saving operation process.

Framework Nucleic Acid Immune Adjuvant for Transdermal Delivery Based Chemo-immunotherapy for Malignant Melanoma Treatment.

Despite the tremendous progresses of cancer immunotherapy, its current clinical responses rate in melanoma remains to be improved. Here, we have reported a skin penetrating tetrahedral framework nucleic acid immune adjuvant (FNAIA) to transdermally deliver chemotherapy drugs into melanoma to induce the immunogenic death of tumor cells and expose tumor antigens, which with assistance of CpG oligodeoxynucleotide incorporated in FNAIA could trigger systemic tumor-specific immune responses. Compared with free CpG, FNAIA could penetrate deeper into subcutaneous tumor tissues and more effectively stimulate dendritic cell maturation. Notably, doxorubicin-loaded FNAIA locally applied on the intact skin above the melanoma could effectively inhibit the growth of mouse B16F10 melanoma and increase tumor CD8+ T cell infiltration. Moreover, combined with immune checkpoint inhibitor, the growth of distant tumors could also be effectively inhibited, suggesting that this strategy could induce systemic immune responses. Therefore, this work provides a new idea for non-invasive treatment of skin cancer.

Triple-Probe DNA Framework-Based Transistor for SARS-CoV-2 10-in-1 Pooled Testing.

Accurate and population-scale screening technology is crucial in the control and prevention of COVID-19, such as pooled testing with high overall testing efficiency. Nevertheless, pooled testing faces challenges in sensitivity and specificity due to diluted targets and increased contaminations. Here, we develop a graphene field-effect transistor sensor modified with triple-probe tetrahedral DNA framework (TDF) dimers for 10-in-1 pooled testing of SARS-CoV-2 RNA. The synergy effect of triple probes as well as the special nanostructure achieve a higher binding affinity, faster response, and better specificity. The detectable concentration reaches 0.025-0.05 copy µL-1 in unamplified samples, lower than that of the reverse transcript-polymerase chain reaction. Without a requirement of nucleic-acid amplification, the sensors identify all of the 14 positive cases in 30 nasopharyngeal swabs within an average diagnosis time of 74 s. Unamplified 10-in-1 pooled testing enabled by the triple-probe TDF dimer sensor has great potential in the screening of COVID-19 and other epidemic diseases.

Split G-Quadruplexes Enhance Nanopore Signals for Simultaneous Identification of Multiple Nucleic Acids.

Assembly of DNA structures based on hybridization like split G-quadruplex (GQ) have great potential for the base-pair specific identification of nucleic acid targets. Herein, we combine multiple split G-quadruplex (GQ) assemblies on designed DNA nanostructures (carrier) with a solid-state nanopore sensing platform. The split GQ probes recognize various nucleic acid sequences in a parallel assay that is based on glass nanopore analysis of molecular structures. Specifically, we split a GQ into two asymmetric parts extended with sequences complementary to the target. The longer G-segment is in solution, and the shorter one is on a DNA carrier. If the target is present, the two separate GQ parts will be brought together to facilitate the split GQ formation and enhance the nanopore signal. We demonstrated detection of multiple target sequences from different viruses with low crosstalk. Given the programmability of this DNA based nanopore sensing platform, it is promising in biosensing.

Controlled Assembly of Fluorophores inside a Nanoliposome.

Cellular compartmentalization plays an essential role in organizing the complex and multiple biochemical reactions in the cell. An artificial compartment would provide powerful strategies to develop new biochemical tools for material production and diagnosis, but it is still a great challenge to synthesize the compartments that encapsulate materials of interest while controlling their accurate locations, numbers, and stoichiometry. In this study, we evaluated chemical characteristics of a liposome-encapsulated compartment, which has great potential to locate various materials of interest with precise control of their locations and numbers in the compartment. A nanoliposome was constructed inside a ring-shaped DNA origami skeleton according to the method of Yang et al., and further equipped with a double-stranded DNA platform to assemble molecules of interest in the nanoliposome. Upon formation of the nanoliposome, a pH-sensitive fluorophore on the bridged platform showed little or no response to the pH change of the outer buffer, ensuring that the molecules assembled on the platform are effectively shielded from the outer environment. The ring-shaped DNA skeleton equipped with a double-stranded DNA platform allows spatial assembly of several functional molecules inside the nanoliposome to isolate them from the outer environment.

Self-assembly Induced Enhanced Electrochemiluminescence of Copper Nanoclusters Using DNA Nanoribbon Templates.

Copper nanoclusters (CuNCs) are attractive electrochemiluminescence (ECL) emitters as Cu is comparatively inexpensive, nontoxic, and highly abundant. However, their ECL yield is relatively low. Herein, we report that orderly self-assembly of CuNCs using DNA nanoribbon as the template (DNR/CuNCs) conferred the CuNCs with improved ECL properties compared with individual CuNCs in both annihilation and co-reactant processes. The DNR/CuNCs resulted in a high ECL yield of 46.8 % in K2 S2 O8 , which was ≈68 times higher than that of individual CuNCs. This strategy was successfully extended to other ECL emitters, such as gold nanoclusters and the Ru(bpy)3 2+ /TPrA system. Furthermore, as an application of DNR/CuNCs, a DNR/CuNC-based ECL biosensor with higher sensitivity was constructed for dopamine determination (two orders of magnitude lower than that previously reported), showing that DNR/CuNCs have a potential for application in ECL bioanalysis as a new type of superior luminophore candidate.

Recent Advances in DNA-Based Cell Surface Engineering for Biological Applications.

Due to its excellent programmability and biocompatibility, DNA molecule has unique advantages in cell surface engineering. Recent progresses provide a reliable and feasible way to engineer cell surfaces with diverse DNA molecules and DNA nanostructures. The abundant form of DNA nanostructures has greatly expanded the toolbox of DNA-based cell surface engineering and gave rise to a variety of novel and fascinating applications. In this review, we summarize recent advances in DNA-based cell surface engineering and its biological applications. We first introduce some widely used methods of immobilizing DNA molecules on cell surfaces and their application features. Then we discuss the approaches of employing DNA nanostructures and dynamic DNA nanotechnology as elements for creating functional cell surfaces. Finally, we review the extensive biological applications of DNA-based cell surface engineering and discuss the challenges and prospects of DNA-based cell surface engineering.

Self-assembled DNA nanostructure containing oncogenic miRNA-mediated cell proliferation by downregulation of FOXO1 expression.

FOXO1 transcription factor not only limits the cell cycle progression but also promotes cell death as a tumor suppressor protein. Though the expression of FOXO1 is largely examined in breast cancer, the regulation of FOXO1 by miRNA is yet to be explored. In the current study, self-assembled branched DNA (bDNA) nanostructures containing oncogenic miRNAs were designed and transfected to the MCF7 cell line to decipher the FOXO1 expression. bDNA containing oncogenic miRNAs 27a, 96, and 182 synergistically downregulate the expression of FOXO1 in MCF7 cells. The down-regulation is evident both in mRNA and protein levels suggesting that bDNA having miRNA sequences can selectively bind to mRNA and inhibit translation. Secondly, the downstream gene expression of p21 and p27 was also significantly downregulated in presence of miR-bDNA nanostructures. The cell proliferation activity was progressively increased in presence of miR-bDNA nanostructures which confirms the reduced tumor suppression activity of FOXO1 and the downstream gene expression. This finding can be explored to design novel bDNA structures which can downregulate the tumor suppressor proteins in normal cells and induce cell proliferation activity to identify early-phase markers of cancer.

Applications of triplex DNA nanostructures in sensor development.

Triplex DNA nanostructures are one of the most emerging and fascinating self-assembled nanostructures due to their unique nanoparticle-like organization and inherit characteristics. They have attracted numerous interests recently because of their versatile and powerful utility in diverse areas of science and technology, such as clinical or disease diagnosis and stimuli-based drug delivery. This review addresses particularly the utilization of DNA triplexes in the development of biosensors for detecting nucleic acid; strategies in sensing pH, protein activity, ions, or molecules. Finally, an outlook for potential applications of triplex DNA nanoswitches is provided.