Y-shaped DNA Structure Research Articles

In Situ Intracellular Autocatalytic Hairpin Assembly of the Y-Shaped DNA Nanostructure for miR-155 Sensing and Gene Silencing.

miR-155 is a class of cancer markers closely related to cancer metastasis and invasion. Combining in situ detection with gene silencing not only helps to analyze the information on the abundance and spatial location of microRNA expression in the cell but also synergizes the therapy. In this work, we prepared HD@CM vesicles with three hairpin DNAs by using MCF-7 cell membranes. The hairpin DNAs can be triggered by endogenous miR-155, which opens the autocatalytic molecular circuit (ACHA) and obtains Y-shaped DNA nanostructures. This nanostructure not only detects endogenous miR-155 with high sensitivity for in situ imaging but also enables gene regulation of intracellular survivin mRNA. The levels of miR-155 in MDA-MB-231, MCF-7, Hela, and HEK-293T cells are found to be 7703, 3978, 1696, and 1229 copies/cell, respectively, as detected by HD@CMs. The fluorescence produced by HD@CM after coincubation with different cells is found to be proportional to the intracellular miR-155 content by confocal imaging. In addition, the gene regulatory function of the Y-shaped DNA structure resulted in significant inhibition of survivin protein expression and apoptosis rates of up to 83%. We look forward to the future application of our HD@CM platform for the precise diagnosis and programmable treatment of clinical cancers.

A controllable Y-shaped DNA structure assisted aptasensor for the simultaneous detection of AFB1 and OTA based on ARGET ATRP.

The development of a simple, rapid, and sensitive technology for the simultaneous detection of mycotoxins is of great significance in ensuring the safety of foods and drugs. Herein, a fluorescence aptasensor with high sensitivity and reproducibility for the simultaneous detection of aflatoxin B1 (AFB1) and ochratoxin A (OTA) was developed. In this sensing system, AFB1 and OTA aptamers were co-immobilized on the surface of magnetic beads (MBs) to form a Y-shaped structure through the principle of complementary base pairing, and were used as recognition probes to specifically capture the target. Activators regenerated by electron transfer for atom transfer radical polymerization (ARGET ATRP) was used as a signal amplification strategy to improve the sensitivity. The initiator modified at the end of an antibody initiates the ARGET ATRP reaction. Different fluorescence signals were designed to achieve the simultaneous detection of OTA and AFB1 with limits of 426.18 and 79.55 fg mL-1 for AFB1 and OTA, respectively. In addition, experiments were conducted on three types of samples, and the recoveries of the two mycotoxins ranged from 87.30% to 109.50%, with relative standard deviations ranging from 0.50% to 4.92% under reproducible conditions. The results suggest that the developed aptasensor is sufficient to meet the different regulatory requirements of the two mycotoxins in food and drug safety and shows great potential.

An NIR light-responsive “on-off-on” photoelectrochemical aptasensor for carcinoembryonic antigen assay based on Y-shaped DNA

This work develops a novel photoelectrochemical sensor for the detection of carcinoembryonic antigen (CEA) based on the composite of UCNPs with semiconductors and conformational changes in the DNA structure. Firstly, SnS2, ZnIn2S4 and UCNPs were assembled on the surface of the ITO electrode. Then Au NPs were dropped, which could facilitate the coupling of CdSe NPs modified DNA1 via Au-S bond, giving an ITO/SnS2/ZnIn2S4/UCNPs/CdSe heterojunction structure. When irradiated with 980nm near-infrared (NIR) light, the UV-visible light emitted by the UCNPs could excite the nanocomposite, producing an enhanced photoelectric reaction. Subsequently, CEA aptamer and DNA2-modified SiO2 were added to form a Y-shaped DNA structure. At this time, the photocurrent was significantly reduced by the combination of the light-blocking effect of SiO2 and the departure of CdSe NPs from the electrode surface. When the target CEA was added, the recognition between CEA and the aptamer led to the collapse of the Y-shaped DNA structure, the restoration of hairpin DNA and the proximity of CdSe to the electrode. Accordingly, the photocurrent signals enhanced again. Under optimal experimental conditions, the detection limit as low as 0.3pgmL-1 was obtained with good selectivity, achieving a sensitive "on-off-on" photoelectrochemical sensor for CEA detection.

A “signal on” photoelectrochemical biosensor for miRNA-21 detection based on layer-by-layer assembled n-n type Bi2S3@CdIn2S4 heterojunction

MicroRNA (miRNA) is an ideal biomarker, and the sensitive monitoring of low-abundance miRNA is of great significance for the treatment of cancer at the onset stage. Herein, a novel Y-shaped DNA-mediated photoelectrochemical (PEC) sensing platform based on the Bi2S3@CdIn2S4 photoanode is proposed for the detection of miRNA-21. First, an n-n type Bi2S3@CdIn2S4 heterojunction with tight interface is formed for the first time through a layer-by-layer assembly strategy due to the matched band structures of Bi2S3 and CdIn2S4. The photocurrent intensity of Bi2S3@CdIn2S4/GCE electrode is 1.7-fold higher than that of Bi2S3/GCE electrode and 2.5-fold higher than that of CdIn2S4/GCE electrode under visible light irradiation. Additionally, miRNA-21-triggered catalytic hairpin assembly facilitates the formation of Y-shaped DNA structure with CdS quantum dots (CdS QDs). As the photosensitizer of Bi2S3@CdIn2S4 heterojunction, CdS QDs accelerate the separation of electron-hole pairs in the sensing system and realizes photocurrent signal enhancement, thus realizing the “signal on” detection of the miRNA-21. The proposed PEC biosensor exhibits a superior sensitivity of 0.18 fM and the recoveries of human serum range from 98.0% to 104% with a relative standard deviation of 5.8–8.9%. Therefore, the constructed PEC biosensor has potential application value in the early diagnosis and treatment of miRNA-related diseases.

CMG helicase disassembly is controlled by replication fork DNA, replisome components and a ubiquitin threshold.

The eukaryotic replisome assembles around the CMG helicase, which stably associates with DNA replication forks throughout elongation. When replication terminates, CMG is ubiquitylated on its Mcm7 subunit and disassembled by the Cdc48/p97 ATPase. Until now, the regulation that restricts CMG ubiquitylation to termination was unknown, as was the mechanism of disassembly. By reconstituting these processes with purified budding yeast proteins, we show that ubiquitylation is tightly repressed throughout elongation by the Y-shaped DNA structure of replication forks. Termination removes the repressive DNA structure, whereupon long K48-linked ubiquitin chains are conjugated to CMG-Mcm7, dependent on multiple replisome components that bind to the ubiquitin ligase SCFDia2. This mechanism pushes CMG beyond a '5-ubiquitin threshold' that is inherent to Cdc48, which specifically unfolds ubiquitylated Mcm7 and thereby disassembles CMG. These findings explain the exquisite regulation of CMG disassembly and provide a general model for the disassembly of ubiquitylated protein complexes by Cdc48.

Simultaneous electrochemical determination of ochratoxin A and fumonisin B1 with an aptasensor based on the use of a Y-shaped DNA structure on gold nanorods.

A complementary DNA (cDNA) was designed to simultaneously hybridize with the ochratoxin A (OTA) aptamer and the fumonisin B1 (FB1) aptamer to form a unique Y-shaped DNA structure and to achieve simultaneous detection. Gold nanorods (AuNRs) were used to immobilize thionine (Th), thiolated ferrocene (Fc), thiolated OTA aptamer (Apt1), and thiolated FB1 aptamer (Apt2), to form an amplified signal element and a recognition element. The Apt1-AuNRs-Th complex and the Apt2-AuNRs-Fc complex hybridize with cDNA to form a unique Y-DNA structure on a gold electrode. This produces two initial electrochemical signals [with 177μΑcm-2 near -0.2V, and 3121μΑcm-2 near +0.46V (vs. Ag/AgCl)] by differential pulse voltammetry. Upon addition of 0.1ngmL-1 OTA and 0.1ngmL-1 FB1, the aptamers bind the two toxins. This results in the release of Apt1-AuNRs-Th and Apt2-AuNRs-Fc, so the peak currents densities decrease to 115μΑcm-2 and 209μΑcm-2. The assay allows simultaneous determination of OTA and FB1 in the 1.0pg·mL-1 to 100ng·mL-1 concentration ranges, with LODs of 0.47 and 0.26pg·mL-1. The assay is reproducible, stable and specific. It was applied to the determination of OTA and FB1 in spiked beer, with recoveries between 89.0% and 102.0%. Graphical abstractSchematic representation of OTA and FB1 detection based on Apt2-AuNRs-Fc/Apt1-AuNRs-Th/cDNA/AuE. (AuNRs: Gold nanorods; Th: thionine; Fc: ferrocene; SH: thiol; BSA: Bovine serum albumin; cDNA: Complementary DNA; Apt1: Aptamer1; Apt2: Aptamer2; OTA: Ochratoxin A; FB1: Fumonisin B1).

Electrochemiluminescence Detection of Silver Ion Based on Trigeminal Structure of DNA

A novel electrochemiluminescence (ECL) biosensor for highly sensitive and selective detection of Ag+ is developed based on the sensitive “turn-on” structure-switching trigeminal structure of deoxyribonucleic acid (DNA-TS). DNA-TS consists of three oligonucleotides hybridized to form three double-stranded DNA like Y-shaped DNA structure. The formation of DNA-TS makes the ECL sign Ru(bpy)2(mcbpy-O-Su-ester)(PF6)2 of reporter probe approach to the electrode surface and thus increase the ECL signal intensity. The “signal-on” ECL biosensor could detect Ag+ concentration in the range from 1.0 × 10-11 to 3.2 × 10-9 mol L-1, and the limit of detection is 3.0 × 10-12 mol L-1. The strategy is sensitive and rapid. This approach could be extended to the design of sensors for other desired heavy metal ions as well. Such ECL sensors are promising for sensitive detection of other heavy metal ions.

A label-free electrochemical biosensor for microRNAs detection based on DNA nanomaterial by coupling with Y-shaped DNA structure and non-linear hybridization chain reaction

DNA nanomaterials have been widely used in bioassays due to their promising properties for sensitive and specific detection of biomolecules. Herein, a label-free electrochemical method was developed for quantitative detection of microRNAs by integrating Y-shaped DNA (Y-DNA) structures with non-linear hybridization chain reaction (non-linear HCR). The Y-DNA structures consisting of three sequences (Y1, Y2 and Y3) serve as stable and specific probes for recognizing target miRNAs. In the presence of target miRNA, competitive hybridization occurs between miRNA and Y-DNA, resulting in the release of Y3 and the disintegration of the Y-DNA structure. The triggers, which were blocked by Y3 previously, were exposed and initiated the non-linear HCR. Remarkable electrochemical signal changes were produced after the isothermal amplification reaction. Therefore, the proposed biosensor achieved sensitive detection of microRNAs (miRNAs). Under optimal conditions, the limit of detection (LOD) was reduced to 0.3334 fM and linear range was from 1 fM to 10 pM. The special design of Y-DNA helped the biosensor obtain the ability to distinguish between single base mutations. What's more, this biosensor was capable of detecting miRNAs in clinical serum samples. We hope that this developed biosensor would provide a potential application for DNA nanomaterials in the field of microRNAs detection and inspire more interests in the development of DNA nanomaterial biosensors.

Turn-off colorimetric sensor for sequence-specific recognition of single-stranded DNA based upon Y-shaped DNA structure

A novel turn-off colorimetric sensor for sequence-specific recognition of single-stranded DNA (ssDNA) was established by combining Y-shaped DNA duplex and G-quadruplex-hemin DNAzyme. A G-rich single-stranded DNA (Oligo-1) displays peroxidase mimicking catalytic activity due to the specific binding with hemin in the presence of K+, which was able to catalyze the oxidation of colorless 2,2′-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS2−) by H2O2 to generate green ABTS•− radical for colorimetric assay. Oligonucleotide 2 (Oligo-2) was partly complementary with Oligo-1 and the target DNA. Upon addition of target DNA, Oligo-1, Oligo-2 and target DNA can hybridize with each other to form Y-shaped DNA duplex. The DNAzyme sequence of Oligo-1 was partly caged into Y-shaped DNA duplex, resulting in the inactivation of the DNAzyme and a sharp decrease of the absorbance of the oxidation product of ABTS2−. Under the optimum condition, the absorbance decreased linearly with the concentration of target DNA over the range of 1.0–250 nM and the detection limit was 0.95 nM (3σ/slope) Moreover, satisfied result was obtained for the discrimination of single-base or two-base mismatched DNA.

Highly Efficient Target Recycling-Based Netlike Y-DNA for Regulation of Electrocatalysis toward Methylene Blue for Sensitive DNA Detection

In this work, the highly efficient target recycling-based netlike Y-shaped DNA (Y-DNA), which regulated the electrocatalysis of Fe3O4@CeO2-Pt nanoparticles (Fe3O4@CeO2-PtNPs) toward methylene blue (MB) for signal amplification, was developed to prepare a sensitive DNA biosensor for detecting the DNA associated with oral cancer. Specifically, with the help of highly efficient enzyme-assisted target recycling (EATR) amplification strategy, one target DNA input was converted to corresponding plenty of DNA strands S1-Fe3O4@CeO2-Pt and S2-MB output, which could be employed to interact with HP2 immobilized on the electrode surface to form stable netlike Y-DNA without any waste of recycling products. Meanwhile, the formation of netlike Y-DNA could regulate electrocatalytic efficiency of Fe3O4@CeO2-PtNPs, inducing the proximity of Fe3O4@CeO2-PtNPs to MB and significantly enhancing electrochemical signal. Further, the signal could also be amplified by Fe3O4@CeO2-PtNPs modified on the electrode surface. By virtue of this ingenious design, a novel netlike Y-DNA structure based on highly efficient EATR was simply constructed and successfully applied to an electrochemical DNA biosensor along with electrocatalysis of Fe3O4@CeO2-PtNPs, achieving the sensitive detection of target DNA ranging from 10 fM to 50 nM with a detection limit of 3.5 fM. Impressively, the biosensor here demonstrates an admirable method for regulating the electrocatalysis of NPs toward substrates to enhance signal, and we believe that this biosensor is a potential candidate for the sensitive detection of target DNA or other disease-related nucleic acids.

A regenerative ratiometric electrochemical biosensor for selective detecting Hg2+ based on Y-shaped/hairpin DNA transformation

Inspired by dual-signaling ratiometric mechanism which could reduce the influence of the environmental change, a novel, convenient, and reliable method for the detection of mercury ions (Hg2+) based on Y-shaped DNA (Y-DNA) was developed. Firstly, the Y-DNA was formed via the simple annealing way of using two different redox probes simultaneously, omitting the multiple operation steps on the electrode. The Y-DNA was immobilized on the gold electrode surface and then an obvious ferrocene (Fc) signal and a weak methylene blue (MB) signal were observed. Upon addition of Hg2+, the Y-DNA structure was transformed to hairpin structure based on the formation of T-Hg2+-T complex. During the transformation, the redox MB gets close to and the redox Fc gets far away from the electrode surface, respectively. This special design allows a reliable Hg2+ detection with a detection range from 1 nM to 5 μM and a low detection limit down to 0.094 nM. Furthermore, this biosensor exhibits good selectivity and repeatability, and can be easily regenerated by using l-cysteine. This study offers a simple and effective method for designing ratiometric biosensors for detecting other ions and biomolecules.

Direct observation of spatial configuration and structural stability of locked Y-shaped DNA structure

A new building block unit (locked Y-DNA) and its structural properties for self-assembled, bottom-up, three-dimensional supramolecular nanoarchitectural probe ​have been introduced using single-molecule FRET imaging.

Membrane-assisted growth of DNA origami nanostructure arrays.

Biological membranes fulfill many important tasks within living organisms. In addition to separating cellular volumes, membranes confine the space available to membrane-associated proteins to two dimensions (2D), which greatly increases their probability to interact with each other and assemble into multiprotein complexes. We here employed two DNA origami structures functionalized with cholesterol moieties as membrane anchors—a three-layered rectangular block and a Y-shaped DNA structure—to mimic membrane-assisted assembly into hierarchical superstructures on supported lipid bilayers and small unilamellar vesicles. As designed, the DNA constructs adhered to the lipid bilayers mediated by the cholesterol anchors and diffused freely in 2D with diffusion coefficients depending on their size and number of cholesterol modifications. Different sets of multimerization oligonucleotides added to bilayer-bound origami block structures induced the growth of either linear polymers or two-dimensional lattices on the membrane. Y-shaped DNA origami structures associated into triskelion homotrimers and further assembled into weakly ordered arrays of hexagons and pentagons, which resembled the geometry of clathrin-coated pits. Our results demonstrate the potential to realize artificial self-assembling systems that mimic the hierarchical formation of polyhedral lattices on cytoplasmic membranes.

Probing Y-shaped DNA structure with time-resolved FRET

Self-assembly based on nucleic acid systems has become highly attractive for bottom-up fabrication of programmable matter due to the highly selective molecular recognition property of biomolecules. In this context, Y-shaped DNA (Y-DNA) provides an effective building block for forming unique self-assembled large-scale architectures. The dimension and growth of the nano- and microstructures depend significantly on the configurational stability of Y-DNA as a building block. Here we present structural studies of Y-DNA systems using a time-resolved FRET (Förster resonance energy transfer) technique. A fluorophore (Alexa 488) and an acceptor (DABCYL) were placed at two different ends of Y-DNA, and the lifetime of the fluorophore was measured to probe the relative distance between the donor and acceptor. Our results confirmed different distances between the arms of the Y-DNA and highlighted the overall structural integrity of the Y-DNA system as a leading building block for molecular self-assembly. Temperature dependent lifetime measurements indicated configurational changes in the overall Y-DNA nanoarchitecture above 40 °C.