Superlarge, Rigidified DNA Tetrahedron with a Y-Shaped Backbone for Organizing Biomolecules Spatially and Maintaining Their Full Bioactivity.

A major impediment to the clinical translation of DNA tiling nanostructures is a technical bottleneck for the programmable assembly of DNA architectures with well-defined local geometry due to the inability to achieve both sufficient structural rigidity and a large framework. In this work, a Y-backbone was inserted into each face to construct a superlarge, sufficiently rigidified tetrahedral DNA nanostructure (called RDT) with extremely high efficiency. In RDT, the spatial size increased by 6.86-fold, and the structural rigidity was enhanced at least 4-fold, contributing to an ∼350-fold improvement in the resistance to nucleolytic degradation even without a protective coating. RDT can be mounted onto an artificial lipid-bilayer membrane with molecular-level precision and well-defined spatial orientation that can be validated using the fluorescence resonance energy transfer (FRET) assay. The spatial orientation of Y-shaped backbone-rigidified RDT is unachievable for conventional DNA polyhedrons and ensures a high level of precision in the geometric positioning of diverse biomolecules with an approximately homogeneous environment. In tests of RDT, surface-confined horseradish peroxidase (HRP) exhibited nearly 100% catalytic activity and targeting aptamer-immobilized gold nanoparticles showed 5.3-fold enhanced cellular internalization. Significantly, RDT exhibited a 27.5-fold enhanced structural stability in a bodily environment and did not induce detectable systemic toxicity.

Spherical Nucleic Acid-Mediated Spatial Matching-Guided Nonenzymatic DNA Circuits for the Prediction and Prevention of Malignant Tumor Invasion.

Detection of intracellular miRNAs, especially sensitive imaging of in vivo miRNAs, is vital to the precise prediction and timely prevention of tumorgenesis but remains a technical challenge in terms of nuclease resistance and signal amplification. Here, we demonstrate a gold nanoparticle-based spherical nucleic acid-mediated spatial matching-guided nonenzymatic DNA circuit (SSDC) for efficient screening of intracellular miRNAs and, in turn, finding cancerous tissues in living organisms before the appearance of clinical symptoms. Due to the substantially enhanced nuclease resistance, the false positive signal is avoided even in a complex biological medium. Target miRNA can straighten out the hairpin DNA probe to be linear, allowing the probe to penetrate into the internal region of a core/shell DNA-functionalized signal nanoampfilier and initiate a strand displacement reaction, generating an amplified fluorescence signal. The detection limit is as low as 17 pM, and miRNA imaging is in good accordance with the gold standard polymerase chain reaction method. The ability to image intracellular miRNAs is substantially superior to that of conventional fluorescence in situ hybridization techniques, making in vivo SSDC-based imaging competent for the precise prediction of tumorigenesis. By intratumoral chemotherapy guided by SSDC-based imaging, tumorigenesis and progression are efficiently controlled before the onset of clinical symptoms.

Y-Shaped Backbone-Rigidified DNA Tiles for the Construction of Supersized Nondeformable Tetrahedrons for Precise Cancer Therapies.

While engineered DNA nanoframeworks have been extensively exploited for delivery of diagnostic and therapeutic regents, DNA tiling-based DNA frameworks amenable to applications in living systems lag much behind. In this contribution, by developing a Y-shaped backbone-based DNA tiling technique, we assemble Y-shaped backbone-rigidified supersized DNA tetrahedrons (RDT) with 100% efficiency for precisely targeted tumor therapy. RDT displays unparalleled rigidness and unmatched resistance to nuclease degradation so that it almost does not deform under the force exerted by the atomic force microscopy tip, and the residual amount is not less than 90% upon incubating in biological media for 24 h, displaying at least 11.6 times enhanced degradation resistance. Without any targeting ligand, RDT enters the cancer cell in a targeted manner, and internalization specificity is up to 15.8. Moreover, 77% of RDT objects remain intact within living cells for 14 h. The drug loading content of RDT is improved by 4-8 times, and RDT almost 100% eliminates the unintended drug leakage in a stimulated physiological medium. Once systemically administrated into HeLa tumor-bearing mouse models, doxorubicin-loaded RDTs preferentially accumulate in tumor sites and efficiently suppress tumor growth without detectable off-target toxicity. The Y-DNA tiling technique offers invaluable insights into the development of structural DNA nanotechnology for precise medicine.

Persistent Targeting DNA Nanocarrier Made of 3D Structural Unit Assembled from Only One Basic Multi-Palindromic Oligonucleotide for Precise Gene Cancer Therapy.

Construction of a simple, reconfigurable, and stimuli-responsive DNA nanocarrier remains a technical challenge. In this contribution, by designing three palindromic fragments, a simplest four-sticky end-contained 3D structural unit (PS-unit) made of two same DNA components is proposed. Via regulating the rotation angle of central longitudinal axis of PS-unit, the oriented assembly of one-component spherical architecture is accomplished with high efficiency. Introduction of an aptamer and sticky tail warehouse into one component creates a size-change-reversible targeted siRNA delivery nanovehicle. Volume swelling of 20 nm allows one carrier to load 1987 siPLK1s. Once entering cancer cells and responding to glutathione (GSH) stimuli, siPLK1s are almost 100% released and original size of nanovehicle is restored, inhibiting the expression of PLK1 protein and substantially suppressing tumor growth (superior to commercial transfection agents) in tumor-bearing mice without systemic toxicity.

Intelligent Reconfiguration-Promoted Cellular Internalization of Core-Shell DNA Nanoprobe Equipped with Successive Dual Stimuli-Responsive Protective Satellites for Amplification Fluorescence Imaging of Tumor Cells.

Although DNA probes have attracted increasing interest for precise tumor cell identification by imaging intracellular biomarkers, the requirement of commercial transfection reagents, limited targeting ligands, and/or non-biocompatible inorganic nanostructures has hampered the clinic translation. To circumvent these shortcomings, a reconfigurable ES-NC (Na+-dependent DNAzyme (E)-based substrate (S) cleavage core/shell DNA nanocluster (NC)) entirely from DNA strands is assembled for precise imaging of cancerous cells in a successive dual-stimuli-responsive manner. This nanoprobe is composed of a strung DNA tetrahedral satellites-based protective (DTP) shell, parallelly aligned target-responsive sensing (PTS) interlayer, and hydrophobic cholesterol-packed innermost layer (HCI core). Tetrahedral axial rotation-activated reconfiguration of DTP shell promotes the exposure of interior hydrophobic moieties, enabling cholesterol-mediated cellular internalization without auxiliary elements. Within cells, over-expressed glutathione triggers the disassembly of the DTP protective shell (first stimulus), facilitating target-stimulated signal transduction/amplification process (second stimuli). Target miRNA-21 is detected down to 10.6 fM without interference from coexisting miRNAs. Compared with transfection reagent-mediated counterpart, ES-NC displays a higher imaging ability, resists nuclease degradation, and has no detectable damage to healthy cells. The blind test demonstrates that the ES-NC is suitable for the identification of cancerous cells from healthy cells, indicating a promising tool for early diagnosis and prediction of cancer.

DNA tetrahedral nanostructures for the biomedical application and spatial orientation of biomolecules.

DNA not only plays a vital role in nature as fundamental hereditary material for storing genetic material, but also serves as well-defined functional material, for example, building blocks for the assembly of nanoscale bio-architectures by Watson-Crick base-pairing interaction. With the development of molecular biology, biotechnology and nanoscience, structural DNA nanotechnology has achieved numerous advances, contributing to the construction of various DNA nanostructures ranging from discrete objects to one dimensional (1D), two dimensional (2D), and three dimensional (3D) architectures. Among them, DNA tetrahedral nanoarchitecture is intensively studied because of simple 3D structure, easy design and unique properties, such as high rigidity, desirable biostability and efficient cellular uptake without auxiliary species. This review summarizes the research progress in the assembly of DNA tetrahedral objects and outlines the applications in biosensing, drug delivery and targeted therapy. Moreover, the dependence of biological activity of biomolecules on DNA tetrahedron-mediated spatially-controlled arrangement and great potential applications are discussed. In addition, the challenges in the design and clinic applications of DNA tetrahedron-based platforms are described, the perspectives towards biomedical applications are foreseen, and our understandings on further studies of DNA tetrahedron are provided, aiming to motivate the development of DNA nanotechnology and interdisciplinary research.

Center backbone-rigidified DNA polygonal nanostructures and bottom face-templated polyhedral pyramids with structural stability in a complex biological medium.

Due to the sequence programmability, good biocompatibility, versatile functionalities and vast sequence space, DNA oligonucleotides are considered to be ideal building blocks for the assembly of diverse nanostructures in one, two and three dimensions that are capable of engineering of multiple functional nucleic acids into a useful tool to implement intended tasks in biological and medical field. However, the construction of wireframe nanostructures consisting of only a few DNA strands remains quite challenging mainly because of the molecular flexibility-based uncontrollability of size and shape. In this contribution, utilizing gel electrophoretic analysis and atomic force microscopy, we demonstrate the modeling assembly technique for the construction of wireframe DNA nanostructures that can be divided into two categories: rigid center backbone-guided modeling (RBM) and bottom face-templated assembly (BTA) that are responsible for the construction of DNA polygons and polyhedral pyramids, respectively. The highest assembly efficiency (AE) is about 100%, while the lowest AE is not less than 50%. Moreover, when adding one edge for polygons or one side face for pyramids, we only need to add one oligonucleotide strand. Especially, the advanced polygons (e.g., pentagon and hexagon) of definite shape are for the first time constructed. Along this line, introduction of cross-linking strands enables the hierarchical assembly of polymer polygons and polymer pyramids. These wireframe DNA nanostructures exhibit the substantially enhanced resistance to nuclease degradation and maintain their structural integrity in fetal bovine serum for several hours even if the vulnerable nicks are not sealed. The proposed modeling assembly technique represents important progress toward the development of DNA nanotechnology and is expected to promote the application of DNA nanostructures in biological and biomedical fields. STATEMENT OF SIGNIFICANCE: DNA oligonucleotides are considered to be ideal building blocks for the assembly of diverse nanostructures. However, the construction of wireframe nanostructures consisting of only a few DNA strands remains quite challenging. In this contribution, we demonstrate the modeling technique for the construction of different wireframe DNA nanostructures: rigid center backbone-guided modeling (RBM) and bottom face-templated assembly (BTA) that are responsible for the assembly of DNA polygons and polyhedral pyramids, respectively. Moreover, cross-linking strands enables the hierarchical assembly of polymer polygons and polymer pyramids. These wireframe DNA nanostructures exhibit the substantially enhanced resistance to nuclease degradation and maintain their structural integrity in fetal bovine serum for several hours, promoting the application of DNA nanostructures in biological and biomedical fields.

Visually predicting microRNA-regulated tumor metastasis by intracellularly 3D counting of fluorescent spots based on in situ growth of DNA flares.

INTRODUCTION: MicroRNAs (miRNAs) have been revealed to be critical genetic regulators in various physiological processes and thus quantitative information on the expression level of critical miRNAs has important implications for the initiation and development of human diseases, including cancers. OBJECTIVES: We herein develop three-dimensionally (3D) counting of intracellular fluorescent spots for accurately evaluating microRNA-21 (miRNA-21) expression in individual HeLa cells based on stimuli-activated in situ growth of optical DNA flares, grid-patterned DNA-protein hybrids (GDPHs). METHODS: Target miRNA is sequence-specifically detected down to 10 pM owing to efficient signal amplification. Within living cells, GDPH flares are nuclease resistant and discrete objects with retarded mobility, enabling the screening of intracellular location and distribution of miRNAs and realizing in situ counting of target species with a high accuracy. RESULTS: The quantitative results of intracellular miRNAs by 3D fluorescence counts are consistent with qPCR gold standard assay, exhibiting the superiority over 2D counts. By screening the expression of intracellular miR-21 that can down-regulate the programmed cell death 4 (PDCD4) protein, the proliferation and migration of HeLa cells, including artificially-regulated ones, were well estimated, thus enabling the prediction of cancer metastasis in murine tumor models. CONCLUSION: The experiments in vitro, ex vivo and in vivo demonstrate that GDPH-based 3D fluorescence counts at the single cell level provide a valuable molecular tool for understanding biological function of miRNAs and especially for recognizing aggressive CTCs, offering a design blueprint for further expansion of DNA structural nanotechnology in predicting distant metastasis and prevention of tumor recurrence after primary resection.

Outward Movement of Targeting Ligands from a Built-In Reserve Pool in Nuclease-Resistant 3D Hierarchical DNA Nanocluster for in Vivo High-Precision Cancer Therapy.

Nanostructures made entirely of DNAs display great potential as chemotherapeutic drug carriers but so far cannot achieve sufficient clinic therapy outcomes due to off-target toxicity. In this contribution, an aptamer-embedded hierarchical DNA nanocluster (Apt-eNC) is constructed as an intelligent carrier for cancer-targeted drug delivery. Specifically, Apt-eNC is designed to have a built-in reserve pool in the interior cavity from which aptamers may move outward to function as needed. When surface aptamers are degraded, ones in reserve pool can move outward to offer the compensation, thereby magically preserving tumor-targeting performance in vivo. Even if withstanding extensive aptamer depletion, Apt-eNC displays a 115-fold enhanced cell targeting compared with traditional counterparts and at least 60-fold improved tumor accumulation. Moreover, one Apt-eNC accommodates 5670 chemotherapeutic agents. As such, when systemically administrated into HeLa tumor-bearing BALB/c nude mouse model, drug-loaded Apt-eNC significantly inhibits tumor growth without systemic toxicity, holding great promise for high precision therapy.

Endogenous Stimuli-Responsive Autonomous Separation of Dual-Targeting DNA Guided Missile from Nanospacecraft for Intelligent Targeted Cancer Therapy.

Most conventional chemotherapeutics indiscriminately kill both cancerous and healthy cells and cause toxic side effects, limiting the maximum tolerated dose and thereby compromising therapeutic efficacy. To address this challenge, here dual-targeting intelligent DNA guided missile (GM)-integrated nanospacecraft (NSC) (abbreviated as GM-NSC) is demonstrated for staged chemotherapeutic drug delivery exclusively into cancer cells and then mitochondria (not into healthy cells). GM-NSC is essentially a core/shell nanocomposite composed of gold nanoparticles (AuNPs) surrounded by a high-density multilayer DNA crown that is self-assembled from DNA tetrahedral units (DNA Tetra) in a highly ordered manner. Each tetrahedral structural unit is equipped with three functional components: a cancer cell-targeting aptamer pointing toward the outside environment, a hidden mitochondria-targeting triphenylphosphonium (TPP), and an explosive bolt (E-bolt). GM-NSC can remain intact in fetal bovine serum solution over 12 h and has 53-fold improved systemic stability. Each GM-NSC accommodates 1250 anticancer doxorubicin (Dox), achieving a 48-63-fold improved drug payload capacity. When systemically administrated into a tumor-bearing xenograft murine model, Dox-loaded GM-NSC enters into tumor sites with 18-fold improved specificity followed by autonomous separation of GMs from the NSC core and specific mitochondrial accumulation due to the explosion of E-bolt upon stimuli of endogenous miRNAs. About 80% of Dox uptaken is transferred into mitochondria and induces mitochondria-mediated apoptosis. As a result, the growth of malignant tumor is almost 100% inhibited without detectable toxicity to healthy tissues. Due to the desirable systemic stability, good biocompatibility, high cargo loading capability, satisfactory in vivo biodistribution, and therapeutic efficacy without adverse effects, intelligible GM-NSC is expected to become an alternative drug delivery system for precision cancer therapy.

Hairpin-inserted cross-shaped DNA nanoprobe for ultrasensitive microRNA detection based on built-in target analogue cycle amplification.

It remains technically challenging to develop a sensitive assay system to isothermally amplify the signal for miRNA detection because of its low abundance in tested sample, sequence similarities and existence in complex biological environments. In this study, using miRNA-21 as target model, a hairpin-inserted cross-shaped DNA nanoprobe (CP) with four functional arms is constructed for the ultrasensitive detection of miRNA via one-step built-in target analogue (BTA) cycle-mediated signal amplification. BTA is pre-locked in one arm of CP probe and inactive. In the presence of target miRNA, BTA can be unlocked and initiate an isothermal amplification process. Utilizing as-designed CP probe, miRNA-21 can be detected to down to 500 fM, and the linear response range spans over five orders of magnitude. The nonspecific signal is less than 1% upon nontarget miRNAs. CP probe exhibits âˆ¼six times enhancement in resistance to nuclease degradation and no obvious degradation-induced fluorescence change is detected during the assay period. The recovery yield ranges from 98.2～105.5% in FBS solution. Because of the high sensitivity, desirable specificity, strong anti-interference ability and substantial increase in nuclease resistance, CP probe is a promising tool for the detection of miRNAs in a complex biological milieu.

Design and application of DNA nanostructures for organelle-targeted delivery of anticancer drugs.

INTRODUCTION: DNA nanostructures targeting organelles are of great significance for the early diagnosis and precise therapy of human cancers. This review is expected to promote the development of DNA nanostructure-based cancer treatment with organelle-level precision in the future. AREAS COVERED: In this review, we introduce the different principles for targeting organelles, summarize the progresses in the development of organelle-targeting DNA nanostructures, highlight their advantages and applications in disease treatment, and discuss current challenges and future prospects. EXPERT OPINION: Accurate targeting is a basic problem for effective cancer treatment. However, current DNA nanostructures cannot meet the actual needs. Targeting specific organelles is expected to further improve the therapeutic effect and overcome tumor cell resistance, thereby holding great practical significance for tumor treatment in the clinic. With the deepening of the research on the molecular mechanism of disease development, especially on tumorigenesis and tumor progression, and increasing understanding of the behavior of biological materials in living cells, more versatile DNA nanostructures will be constructed to target subcellular organelles for drug delivery, essentially promoting the early diagnosis of cancers, classification, precise therapy and the estimation of prognosis in the future.

A sensing system constructed by combining a structure-switchable molecular beacon with nicking-enhanced rolling circle amplification for highly sensitive miRNA detection.

The detection of disease-related biomarkers, including microRNA (miRNA), is of crucial importance in reducing the morbidity and mortality of cancer. Thus, there is a great desire to develop an efficient and simple sensing method to fulfill the detection of miRNAs. In this study, a novel amplification assay strategy is demonstrated for the highly sensitive detection of miRNA-21 by combining a structure-switchable molecular beacon with nicking-enhanced rolling circle amplification (SMB-NRCA). A circular padlock probe (CPP) contains a target recognition sequence, two binding sites for nicking endonuclease and three hybridization sites for SMBs. miRNA-21 can hybridize with the CPP and act as polymerization primer that initiates the rolling circle amplification (RCA) reaction and two different nicking-mediated RCA processes, releasing a large amount of SMBs and leading to a significantly amplified fluorescence signal originating from the restoration of pre-quenched fluorescence via their structural switching. Via the signal amplification based on the combination of RCA, nicking and SDA, this assay system can quantitatively detect miRNA-21 in a linear change of three orders of magnitude with a detection limit of 1 pM. The assay specificity is very high so that there is no interference from coexisting miRNAs. Moreover, the sensing system possesses ideal anti-interference ability in complicated milieux such as human serum. The novel sensing strategy shows tremendous prospects for application in tumor diagnosis and clinical therapy guidance.

Structure-switchable aptamer-arranged reconfigurable DNA nanonetworks for targeted cancer therapy.

The structural DNA nanotechnology holds great potential application in bioimaging, drug delivery and cancer therapy. Herein, an intelligent aptamer-incorporated DNA nanonetwork (Apt-Nnes) is demonstrated for cancer cell imaging and targeted drug delivery, which essentially is a micron-scale pattern with the thickness of double-stranded monolayer. Cancer cell-surface receptors can make it perform magical transformation into small size of nanosheet intermediates and specifically enter target cells. The binding affinity of Apt-Nnes is increased by 3-fold due to multivalent binding effect of aptamers and it can maintain the structural integrity in fetal bovine serum (FBS) for 8 h. More interestingly, target cancer cells can cause the structural disassembly, and each resulting unit transports 4963 doxorubicin (Dox) into target cells, causing the specific cellular cytotoxicity. The cell surface receptor-mediated disassembly of large size of DNA nanostructures into small size of fractions provides a valuable insight into developing intelligent DNA nanostructure suitable for biomedical applications.

Nonenzymatic Autonomous Assembly of Cross-Linked Network Structures from Only Two Palindromic DNA Components for Intracellular Fluorescence Imaging of miRNAs.

The abnormal expression of miRNA-21 is often found in tumor specimens and cell lines, and thus, its specific detection is an urgent need for the diagnosis and effective therapy of cancers. In this contribution, we demonstrate a palindrome-based hybridization chain reaction (PHCR) upon the stimuli of a short oligonucleotide trigger to perform the autonomous assembly of cross-linked network structures (CNSs) for the amplification detection of miRNA-21 and sensitive fluorescence imaging of cancerous cells. The building blocks are only two palindromic hairpin-type DNA strands that are separately modified with different fluorophores (Cy3 and Cy5), which is easily combined with the catalytic hairpin assembly (CHA) technique that can further amplify the signal output. Utilizing the CHA-PHCR assay system, a small amount of miRNA-21 can activate many triggers via CHA and in turn induce the PHCR-based CNS assembly from more DNA building blocks, bringing Cy3 and Cy5 into close proximity to each other and generating ultrasensitive fluorescence resonance energy transfer signals. As a result, target miRNA can be quantitatively detected down to as low as 10 pM with high assay specificity. The coexisting nontarget miRNAs and other biomacromolecules do not interfere with signal transduction. The developed assay system is suitable for screening different expression levels of miRNA-21 in living cells by fluorescence imaging. The palindrome-based cross-linking assembly can enhance the intracellular stability of assembled nanostructures by at least fivefold and exhibit the good universality for the detection of other miRNAs. Moreover, cancerous cells can be distinguished from healthy cells, and the CHA-PHCR assay is in good accordance with the gold standard PCR method, indicating a promising platform for the diagnosis of human cancers and other genetic diseases.

Swelling of Serum-Stable DNA Nanoparticles upon Target-Induced Conformational Rearrangement of Sensing Probes for the Signal-On Detection of Cancer-Related Genes.

Nuclease-resistant assay probes are of significant importance for biochemical analysis and disease diagnosis. In this contribution, a reconfigurable lipidic moiety-attached DNA nanoparticle (LDN) is constructed from a cholesterol-conjugated multifunctional hairpin-type DNA probe (Chol-DP) by hydrophobicity-mediated self-assembly. The LDN holds high serum stability and displays a low false-positive signal even in a complex biological milieu. The hydrophobic cholesterol moiety enables the hydrophobicity-mediated assembly, while hydrophilic DNA sequence serves as a recognition element and a polymerization template. The initiator-activated strand displacement amplification (SDA) reaction can convert the hairpin-shaped probe into rigid double-stranded DNA (dsDNA), causing the conformational rearrangement-based LDN swelling that can be used to reliably and fluorescently signal the cancer-related p53 gene. The size increase and structural reconfiguration are confirmed by dynamic light scattering (DLS) analysis and confocal microscopy imaging, respectively. Target p53 is specifically detected down to 10 pM. The whole assay process involved only several simple mixing steps. Recovery test and blind test further confirm the feasibility of the use of the LDN for the detection of target DNA in a complex biological milieu, indicating a promising nanotool for biomedical applications.

Tumor-targeting [2]catenane-based grid-patterned periodic DNA monolayer array for in vivo theranostic application.

DNA nanotechnology is often used to build various nano-structures for signaling and/or drug delivery, but it essentially suffers from several major limitations, such as a large number of DNA strands and limited targeting ligands. Moreover, there is no report on in vivo two-dimensional DNA arrays because of various technical challenges. By cross-catenating two palindromic DNA rings, herein, we demonstrate a catenane-based grid-patterned periodic DNA monolayer array ([2]GDA) capable of preferentially accumulating in tumor tissues without any targeting ligands, with a thickness equal to the double-helical DNA monolayer (nearly 2 nm). The structural flexibility of [2]GDA enabled it to fold into a spherical object in solution, favoring cellular uptake. Thus, its cellular internalization activity was comparable with that of the commercial lipofectamine 3000. Moreover, [2]GDA retained the structural integrity over 24 h incubation in biological solutions, achieving a 360-fold improvement in in vivo stability. Significantly, anticancer drug-loaded [2]GDA exhibits desirable therapeutic efficacy in tumor-bearing animals without detectable side effects.

Ultrasensitive Electrochemical Detection of cancer-Related Point Mutations Based on Surface-Initiated Three-Dimensionally Self-Assembled DNA Nanostructures from Only Two Palindromic Probes.

Sensitive and selective detection of proto-oncogenes, especially recognition of point mutation, is of great importance in cancer diagnosis. Here, a ligation-mediated technique is demonstrated for the construction of an intertwined three-dimensional DNA nanosheet (3D SDN) on an electrode surface from only two palindromic hairpin probes (HP1 and HP2), creating a powerful electrochemical biosensor (E-biosensor) for the detection of the p53 gene. First, a capturing probe (CP) is immobilized on an electrode surface via Au-S chemistry, forming an electrochemical sensing interface. In the presence of the target p53 (T), the triggering probe is covalently linked to CP by a ligase. Moreover, target hybridization/ligation/dehybridization process is repeated, amplifying the target hybridization event and increasing the content of surface-confined triggering fragments. As a result, HP1 is opened and in turn interacts with HP2, forming intertwined 3D SDN where HP1 and HP2 are alternately arranged in parallel. Common hybridization and interaction between palindromic fragments are responsible for the assembly in the horizontal and vertical directions, respectively. An electrochemical indicator, methylene blue (MB), can be inserted into 3D SDN, generating a strong electrochemical signal. Utilizing the 3D SDN-based E-biosensor, the target DNA is detected down to 3 fM with a linear response range from 10 fM to 10 nM. Single point mutations are reliably identified even in fetal bovine serum and cellular homogenate. Because of the several advantages of simple design, good universality, inexpensive instrumentation, high assay specificity, and sensitivity, the 3D SDN-based E-biosensor is expected to provide a potential platform for screening point mutation required by early clinical diagnostics and medical research.

Self-Protected DNAzyme Walker with a Circular Bulging DNA Shield for Amplified Imaging of miRNAs in Living Cells and Mice.

Abnormal expression of miRNAs is often detected in various human cancers. DNAzyme machines combined with gold nanoparticles (AuNPs) hold promise for detecting specific miRNAs in living cells but show short circulation time due to the fragility of catalytic core. Using miRNA-21 as the model target, by introducing a circular bulging DNA shield into the middle of the catalytic core, we report herein a self-protected DNAzyme (E) walker capable of fully stepping on the substrate (S)-modified AuNP for imaging intracellular miRNAs. The DNAzyme walker exhibits 5-fold enhanced serum resistance and more than 8-fold enhanced catalytic activity, contributing to the capability to image miRNAs much higher than commercial transfection reagent and well-known FISH technique. Diseased cells can accurately be distinguished from healthy cells. Due to its universality, DNAzyme walker can be extended for imaging other miRNAs only by changing target binding domain, indicating a promising tool for cancer diagnosis and prognosis.

Stimuli-Induced Upgrade of Nuclease-Resistant DNA Nanostructure Composed of a Single Molecular Beacon for Detecting Mutant Genes.

As a kind of cell-free DNA in the bloodstream liberated from tumor cells, circulating tumor DNAs (ctDNAs) have been recognized as promising biomarkers in the field of early cancer diagnosis. However, robust, sensitive, and accurate detection of ctDNA in serum remains extremely challenging, especially toward the mutant KRAS gene, one of the most frequently mutated genes. Although DNA oligonucleotides as emerging practical signaling materials have been developed as sensitive and accurate tools, some intrinsic defects need to be overcome, such as fragility in complex biological environments. In this work, on the basis of the hydrophilicity-promoted assembly, a core/shell DNA nanostructure (DNS-MB) probe is constructed from only one hairpin-shaped probe (cholesterol-modified palindromic molecular beacon, Chol-PMB) for the amplification detection of KRAS mutation in serum without the need for any auxiliary probe. Chol-PMB is designed to recognize target DNA and serve as a polymerization primer and template, and thus target species can initiate polymerization-based strand displacement amplification (SDA). Moreover, target DNA is able to induce further aggregation of DNS-MB particles due to the enzymatic cross-linking effect, leading to a structural upgrade. The DNS-MB probe exhibits a detection limit of 50 fM and a wide quantitative range (from 50 fM to 160 nM). In addition, single nucleotide polymorphisms can be discriminated, such as mutant KRAS G12D (KRAS-M), providing a desirable platform for screening ctDNAs. More excitingly, because the termini of DNA components are hidden inward from nuclease attack, DNS-MB circumvents a false-positive signal even in freshly sampled serum and is suitable for application in the complex biological milieu. As a proof of concept, the DNS-MB probe is expected to provide useful insight into the development of simple and degradation-resistant DNA probes for substantially amplified detection of ctDNAs in complex serum, showing potential applications in the field of early tumor diagnosis.