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Nucleic acids are considered as promising materials for developing exquisite nanostructures from one to three dimensions. The advances of DNA nanotechnology facilitate ingenious design of DNA nanostructures with diverse shapes and sizes. Especially, the algebraic topological framework nucleic acids (ATFNAs) are functional DNA nanostructures that engineer guest molecules (e. g., nucleic acids, proteins, small molecules, and nanoparticles) stoichiometrically and spatially. The intrinsic precise properties and tailorable functionalities of ATFNAs hold great promise for biological applications, such as cell recognition and immunotherapy. This Perspective highlights the concept and development of precisely assembled ATFNAs, and outlines the new frontiers and opportunities for exploiting the structural advantages of ATFNAs for biological applications., (© 2024 Wiley-VCH GmbH.)

The trans-cleavage property of CRISPR-Cas12a system makes it an excellent tool for disease diagnosis. Nevertheless, most methods based on CRISPR-Cas system still require pre-amplification of the target to achieve the desired detection sensitivity. Here we generate Framework-Hotspot reporters (FHRs) with different local densities to investigate their effect on trans-cleavage activity of Cas12a. We find that the cleavage efficiency increases and the cleavage rate accelerates with increasing reporter density. We further construct a modular sensing platform with CRISPR-Cas12a-based target recognition and FHR-based signal transduction. Encouragingly, this modular platform enables sensitive (100 fM) and rapid (<15 min) detection of pathogen nucleic acids without pre-amplification, as well as detection of tumor protein markers in clinical samples. The design provides a facile strategy for enhanced trans cleavage of Cas12a, which accelerates and broadens its applications in biosensing., (© 2023 Wiley-VCH GmbH.)

The precise regulation of interactions of specific immunological components is crucial for controllable immunomodulation, yet it remains a great challenge. With the assistance of advanced computer design, programmable nucleic acid nanotechnology enables the customization of synthetic nucleic acid nanodevices with unprecedented geometrical and functional precision, which have shown promising potential for precise immunoengineering. Notably, the inherently immunologic functions of nucleic acids endow these nucleic acid-based assemblies with innate advantages in immunomodulatory engagement. In this review, the roles of nucleic acids in innate immunity are discussed, focusing on the definition, immunologic modularity, and enhanced bioavailability of structural nucleic acid nanodevices. In light of this, molecular programming and precise organization of functional modules with nucleic acid nanodevices for immunomodulation are emphatically reviewed. At last, the present challenges and future perspectives of nucleic acid nanodevices for immunomodulation are discussed., (© 2023 Wiley-VCH GmbH.)

Designing an ocular drugs delivery system that can permeate the outer blood-retinal barrier (oBRB) is crucial for the microinvasive or noninvasive treatment of ocular fundus diseases. However, due to the lack of a nanocarrier that can maintain structure and composition at the oBRB, only intravitreal injection at the eyeball can deliver therapeutics directly to the ocular fundus via paracellular and intercellular routes, despite the intraocular operations risks. Here, we demonstrated tetrahedral framework nucleic acids (tFNAs) can penetrate the oBRB and deliver therapeutic nucleic acids to the retina of the rat eye in vivo following subconjunctival injection. We also discovered that tFNAs were transported via a paracellular route across the intercellular tight junctions at the oBRB. The histology analysis for ocular layers indicated that individual and aptamer/doxorubicin-loaded tFNAs penetrated all layers of the posterior segment of the eyeball to reach the innermost retina and persisted for over 3 days with minimal systemic biodistribution. We expect that the programmability and penetrability of tFNAs will provide a promising method for drug delivery across oBRB and long-term sustenance at the target site via periocular administration to various tissues.

The ability to design nanostructures with arbitrary shapes and controllable motions has made DNA nanomaterials used widely to construct diverse nanomachines with various structures and functions. The DNA nanostructures exhibit excellent properties, including programmability, stability, biocompatibility, and can be modified with different functional groups. Among these nanoscale architectures, DNA walker is one of the most popular nanodevices with ingenious design and flexible function. In the past several years, DNA walkers have made amazing progress ranging from structural design to biological applications including constructing biosensors for the detection of cancer-associated biomarkers. In this review, the key driving forces of DNA walkers are first summarized. Then, the DNA walkers with different numbers of legs are introduced. Furthermore, the biosensing applications of DNA walkers including the detection- of nucleic acids, proteins, ions, and bacteria are summarized. Finally, the new frontiers and opportunities for developing DNA walker-based biosensors are discussed., (© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Molecular transport controls the efficiency of complex biological network systems such as cellular signaling system and cascade biomedical reaction. However, device fabrication for molecular sensing is often restricted by a low transport efficiency and complicated processing. Here, we report a molecular threading-dependent transport system using three-dimensional (3D) paper origami enabling the directional transport of biomolecules. We demonstrate that framework nucleic acid-based interface engineering allows orthogonal molecular recognition and enzymatic reaction with programmed order on site. We thus develop a single-step electrochemical DNA sensor for quantitative analysis with 1 picomolar sensitivity within 60 min. Our sensor can discriminate a mismatched target at the level of a single base mismatch. Our study shows a great potential toward the development of a biomimetic molecular transport system for point-of-care and precision diagnosis.

Weak ligand-receptor recognition events are often amplified by recruiting multiple regulatory biomolecules to the action site in biological systems. However, signal amplification in in vitro biomimetic systems generally lack the spatiotemporal regulation in vivo. Herein we report a framework nucleic acid (FNA)-programmed strategy to develop valence-controlled signal amplifiers with high modularity for ultrasensitive biosensing. We demonstrated that the FNA-programmed signal amplifiers could recruit nucleic acids, proteins, and inorganic nanoparticles in a stoichiometric manner. The valence-controlled signal amplifier enhanced the quantification ability of electrochemical biosensors, and enabled ultrasensitive detection of tumor-relevant circulating free DNA (cfDNA) with sensitivity enhancement of 3-5 orders of magnitude and improved dynamic range., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The occurrence and prognosis of many complex diseases, such as cancers, is associated with the variation of various molecules, including DNA at the genetic level, RNA at the regulatory level, proteins at the functional level and small molecules at the metabolic level (defined collectively as multilevel molecules). Thus it is highly desirable to develop a single platform for detecting multilevel biomarkers for early-stage diagnosis. Here we report a protocol on DNA-nanostructure-based programmable engineering of the biomolecular recognition interface, which provides a universal electrochemical biosensing platform for the ultrasensitive detection of nucleic acids (DNA/RNA), proteins, small molecules and whole cells. The protocol starts with the synthesis of a series of differentially sized, self-assembled tetrahedral DNA nanostructures (TDNs) with site-specifically modified thiol groups that can be readily anchored on the surface of a gold electrode with high reproducibility. By exploiting the rigid structure, nanoscale addressability and versatile functionality of TDNs, one can tailor the type of biomolecular probes appended on individual TDNs for the detection of specific molecules of interest. Target binding occurring on the gold surface patterned with TDNs is quantitatively translated into electrochemical signals via a coupled enzyme-based catalytic process. This uses a sandwich assay strategy in which biotinylated reporter probes recognize TDN-bound target biomolecules, which then allow binding of horseradish-peroxidase-conjugated avidin (avidin-HRP). Hydrogen peroxide (H2O2) is then reduced by avidin-HRP in the presence of TMB (3,3',5,5'-tetramethylbenzidine) to generate a quantitative electrochemical signal. The time range for the entire protocol is ∼1 d, whereas the detection process takes ∼30 min to 3 h.

Efficient biocatalytic cascade reactions play a crucial role in guiding intricate, specific and selective intracellular transformation processes. However, the catalytic activity of the enzyme cascade reaction in bulk solution was greatly impacted by the spatial morphology and inter‐enzyme distance. The programmability and addressability nature of framework nucleic acid (FNA) allows to be used as scaffold for immobilization and to direct the spatial arrangement of enzyme cascade molecules. Here, we used tetrahedral DNA framework (TDF) as nanorulers to assemble two enzymes for constructing a double‐enzyme complex, which significantly enhance the catalytic efficiency of sarcosine oxidase (SOx)/horseradish peroxidase (HRP) cascade system. We synthesized four types of TDF nanorulers capable of programming the lateral distance between enzymes from 5.67 nm to 12.33 nm. Enzymes were chemical modified by ssDNA while preserving most catalytic activity. Polyacrylamide gel electrophoresis (PAGE), transmission electron microscopy (TEM) and atomic force microscopy (AFM) were used to verify the formation of double‐enzyme complex. Four types of double‐enzyme complexes with different enzyme distance were constructed, in which TDF26(SOx+HRP) exhibited the highest relative enzyme cascade catalytic activity, ~3.11‐fold of free‐state enzyme. Importantly, all the double‐enzyme complexes demonstrate a substantial improvement in enzyme cascade catalytic activity compared to free enzymes. [ABSTRACT FROM AUTHOR]

Nucleic acids in biofluids are emerging biomarkers for the molecular diagnostics of diseases, but their clinical use has been hindered by the lack of sensitive detection assays. Herein, we report the development of a sensitive nucleic acid detection assay named SPOT (sensitive loop‐initiated DNAzyme biosensor for nucleic acid detection) by rationally designing a catalytic DNAzyme of endonuclease capability into a unified one‐stranded allosteric biosensor. SPOT is activated once a nucleic acid target of a specific sequence binds to its allosteric module to enable continuous cleavage of molecular reporters. SPOT provides a highly robust platform for sensitive, convenient and cost‐effective detection of low‐abundance nucleic acids. For clinical validation, we demonstrated that SPOT could detect serum miRNAs for the diagnostics of breast cancer, gastric cancer and prostate cancer. Furthermore, SPOT exhibits potent detection performance over SARS‐CoV‐2 RNA from clinical swabs with high sensitivity and specificity. Finally, SPOT is compatible with point‐of‐care testing modalities such as lateral flow assays. Hence, we envision that SPOT may serve as a robust assay for the sensitive detection of a variety of nucleic acid targets enabling molecular diagnostics in clinics. [ABSTRACT FROM AUTHOR]

Simultaneous and non-destructive quantitative detection of intracellular metal ions holds great promise for improving the accuracy of diagnosis and biological research. Herein, novel multicolor DNAzymes-embedded framework nucleic acids (FNAzymes) were presented, which can easily enter cells and achieve simultaneous and quantitative detection of intracellular physiologically related Cu2+ and Zn2+. Two types of DNAzymes, specific to Cu2+ and Zn2+, were encoded in the framework nucleic acids (FNAs) via self-assembly. With the formation of a well-ordered FNAzyme nanostructure, the fluorophore and the quencher were close to each other; therefore, the fluorescence was quenched. In the presence of Cu2+ and Zn2+, the integrated FNAzymes would be specifically cleaved, resulting in the release of fluorophores in cells. Consequently, the fluorescence in living cells could be observed by a confocal microscope and semi-quantitatively analyzed by flow cytometry with low-nanomolar sensitivity for both metal ions. The FNAzymes have high uniformity and structural accuracy, which are beneficial for intracellular detection with excellent reproducibility. This proposed method offers new opportunities for non-destructive, semi-quantitative, multi-target detection in living cells. [ABSTRACT FROM AUTHOR]

DNA nanostructures are among the most fascinating self-assembled nanostructures in diverse areas of science and technology, because of their nanoscale precision in biomolecule and nanoparticle organization. The implementation of dynamic and spatial regulation in structural morphology and hierarchical assembly upon specific external stimuli will greatly expand their applications in biocomputation, clinical diagnosis, and cancer therapy. Recently, noncanonical nucleic acids, particularly DNA triplexes, i-motifs, and G-quadruplexes, have become powerful tools for biosensing and mechanical switching. Developments in incorporating stimuli-responsive noncanonical nucleic acids into DNA nanostructures provide a promising approach to regulating the spatial organization and hierarchical assembly of DNA nanostructures. In this review, we briefly introduce recent progress in constructing DNA nanostructures with dynamic regulation of the structural transformation and programmable assembly pathways at the nanometer scale by noncanonical nucleic acids and discuss their potential applications and challenges. DNA double helix exploiting Watson–Crick base-pairing lays the foundation of DNA nanotechnology. However, other forms of nucleic acids (e.g., triplex, i-motif, and G-quadruplex) exhibiting noncanonical base-base interactions bring about novel functionality. Here, we review the interplay of naturally occurring noncanonical nucleic acids and artificial DNA nanostructures in biomedical applications that have not been possible by duplex formation alone. [ABSTRACT FROM AUTHOR]

Abstract Biosensor is a device that responds to a particular target in a selective way by incorporation of biological recognition as sensing unit. For practical biomedical applications, a digital output (a qualitative YES/NO answer) is highly demanded for certain end‐user or point‐of‐care applications. Boolean logic, which can be applied to any type of information expressed as 0 (NO) and 1 (YES), is widely employed to achieve such qualitative analysis. Over the past decade, owing to DNA's advantages of stability, accessibility, and manipulability, significant research efforts have been focused on the design and application of DNA logic gates (DLG)‐based biosensors capable of implementing logic‐gated biomedical functions. This review summarizes the existed representative examples and the advanced developments of DLG biosensors, and discusses the limitations and the future directions on the development of novel nanosensors based on DNA logic operations for realizing highly efficient diagnosis. [ABSTRACT FROM AUTHOR]

Aligning carbon nanotubes (CNTs) is a key challenge for fabricating CNT‐based electronic devices. Herein, we report a spherical nucleic acid (SNA) mediated approach for the highly precise alignment of CNTs at prescribed sites on DNA origami. We find that the cooperative DNA hybridization occurring at the interface of SNA and DNA‐coated CNTs leads to an approximately five‐fold improvement of the positioning efficiency. By combining this with the intrinsic positioning addressability of DNA origami, CNTs can be aligned in parallel with an extremely small angular variation of within 10°. Moreover, we demonstrate that the parallel alignment of CNTs prevents incorrect logic functionality originating from stray conducting paths formed by misaligned CNTs. This SNA‐mediated method thus holds great potential for fabricating scalable CNT arrays for nanoelectronics. [ABSTRACT FROM AUTHOR]

Identification of tumor-related mRNA in living cells hold great promise for early cancer diagnosis and pathological research. Herein, we present poly-adenine (polyA)-mediated fluorescent spherical nucleic acid (FSNA) probes for intracellular mRNA detection with regulable sensitivities by programmably adjusting the loading density of DNA on gold nano-interface. Gold nanoparticles (AuNPs) functionalized with polyA-tailed recognition sequences were hybridized to fluorescent “reporter” strands to fabricate fluorescence-quenched FSNA probes. While exposed to target gene, the “reporter” strands were released from FSNA through strand displacement and fluorescence was recovered. With polyA20 tail as the attaching block, the detection limit of FSNA probes was calculated to be 0.31 nM, which is ~55 fold lower than that of thiolated probes without surface density regulation. Quantitative intracellular mRNA detection and imaging could be achieved with polyA-mediated FSNA probes within 2 hours, indicating their application potential in rapid and sensitive intracellular target imaging. [ABSTRACT FROM AUTHOR]

Abstract: Weak ligand–receptor recognition events are often amplified by recruiting multiple regulatory biomolecules to the action site in biological systems. However, signal amplification in in vitro biomimetic systems generally lack the spatiotemporal regulation in vivo. Herein we report a framework nucleic acid (FNA)‐programmed strategy to develop valence‐controlled signal amplifiers with high modularity for ultrasensitive biosensing. We demonstrated that the FNA‐programmed signal amplifiers could recruit nucleic acids, proteins, and inorganic nanoparticles in a stoichiometric manner. The valence‐controlled signal amplifier enhanced the quantification ability of electrochemical biosensors, and enabled ultrasensitive detection of tumor‐relevant circulating free DNA (cfDNA) with sensitivity enhancement of 3–5 orders of magnitude and improved dynamic range. [ABSTRACT FROM AUTHOR]

Precise control over the valency of quantum dots (QDs) is critical and fundamental for quantitative imaging in living cells. However, prior approaches on valence control of QDs remain restricted to single types of valences. A DNA-programmed general strategy is presented for valence engineering of QDs with high modularity and high yield. By employing a series of programmable DNA scaffolds, QDs were generated with tunable valences in a single step with near-quantitative yield (>95 %). The use of these valence-engineered QDs was further demonstrated to develop 12 types of topologically organized QDs-QDs and QDs-AuNPs and 4 types of fluorescent resonance energy transfer (FRET) nanostructures. Quantitative analysis of the FRET nanostructures and live-cell imaging reveal the high potential of these nanoprobes in bioimaging and nanophotonic applications. [ABSTRACT FROM AUTHOR]

DNA-based machines have attracted rapidly growing interest owing to their potential in drug delivery, biocomputing, and diagnostic applications. Herein, we report a type of exonuclease III (Exo III)-powered stochastic DNA walker that can autonomously move on a spherical nucleic acid (SNA)-based 3D track. The motion is propelled by unidirectional Exo III digestion of hybridized DNA tracks in a burnt-bridge mechanism. The operation of this Exo III-propelled DNA walker was monitored in real time and at the single-particle resolution using total internal reflection fluorescence microscopy (TIRF). We further interrogated the morphological effect of the 3D track on the nuclease activity, which suggested that the performance of the DNA walker was critically dependent upon the DNA density and the track conformation. Finally, we demonstrated potential bioanalytical applications of this SNA-based stochastic DNA walker by exploiting movement-triggered cascade signal amplification. [ABSTRACT FROM AUTHOR]

The detection of biomarkers using sensitive and selective analytical devices is critically important for the early stage diagnosis and treatment of diseases. The synergy between the high specificity of nucleic acid recognition units and the great sensitivity of electrochemical signal transductions has already shown promise for the development of efficient biosensing platforms. Yet nucleic-acid based electrochemical biosensors often rely on target amplification strategies (e.g., polymerase chain reactions) to detect analytes at clinically relevant concentration ranges. The complexity and time-consuming nature of these amplification methods impede moving nucleic acid-based electrochemical biosensors from laboratory-based to point-of-care test settings. Fortunately, advancements in nanotechnology have provided growing evidence that the recruitment of nanoscaled materials and structures can enhance the biosensing performance (particularly in terms of sensitivity and response time) to the level suitable for use in point-of-care diagnostic tools. This Review highlights the significant progress in the field of nucleic acid-based electrochemical nanobiosensing with the focus on the works published during the last five years. [ABSTRACT FROM AUTHOR]

Abstract DNA-gold nanoparticles (AuNPs) conjugate is one of the most versatile bionanomaterials for biomedical and clinical diagnosis. However, to finely tune the hybridization ability and precisely control the orientation and conformation of surface-tethered oligonucleotides on AuNPs remains a hurdle. In this work, we developed a poly adenine-mediated spherical nucleic acid (polyA-mediated SNA) strategy by assembling di-block DNA probes on gold nanoparticles (AuNPs) to spatially control interdistance and hybridization ability of oligonucleotides on AuNPs. By modulating length of poly A bound on the SNA with different degrees of constructing, we presented significant improved biosensing performance including high hybridization efficiency, and expanded dynamic range of analytes with more sensitive detection limit. Furthermore, this polyA design could facilitate the programmable detection for DNA in serum environment and simultaneous multicolor detection of three different microRNAs associated with pancreatic carcinoma. The demonstration of the link between modulation of SNA assembly strategy and biodetection capability will increase the development of high performance diagnostic tools for translational biomedicine. Highlights • Poly adenines (polyA)-mediated spherical nucleic acid assembly strategy was developed. • Poly adenines assembly could systematically modulate the lateral spacing and surface density of DNA on AuNPs. • A panel of three pancreatic carcinoma associated miRNAs was simultaneously detected. [ABSTRACT FROM AUTHOR]

Functional imaging of the brain gives us increasingly detailed information about the location of nervous activity. Imaging probes are therefore in demand to target and visualize the neurological processes. However, the deep location of the brain and the blood-brain barrier (BBB) poses challenges in signal detection and probe delivery to the brain. In recent years, nucleic acid imaging probes have been a research frontline to cope with these challenges. Nucleic acid probes showed advantages in preparation, functionalization, targeting and BBB-penetrating. This review will focus on nucleic acid probes-based neuroimaging. In the beginning, common categories of nucleic acid imaging probes are introduced with the targeting strategies. In the next part, we focus on recent advances in strategies of deep tissue imaging in situ and delivery across the blood-brain barrier (BBB). In addition, the perspective on the advantages, limitations and potential development of neural nucleic acid functional imaging probes are elaborated. • Nucleic acids-based probes have realized the specific and smart imaging for the targets or process in neuroscience. • The engineered nucleic acid probes could pass through the blood-brain barrier efficiently and achieve internal tissue imaging. • Nucleic acids served as a powerful imaging platform for imaging different targets in neuroscience in different ways. [ABSTRACT FROM AUTHOR]