DNA nanosensor based on bipedal 3D DNA walker-driven proximal catalytic hairpin assembly for sensitive and fast TK1 mRNA detection.

Hyperproliferative  diseases are the first step for tumor formation; thymidine kinase 1 (TK1) mRNA is closely related to cell proliferation. Therefore, the risk of malignant proliferation can be identified by sensitively detecting the variance in TK1 mRNA concentration, which can be used for tumor auxiliary diagnosis and monitoring tumor treatment. Owing to the low abundance and instability of TK1 mRNA in real samples, the development of a sensitive and fast mRNA detection method is necessary. A DNA nanosensor that can be used for detecting TK1 mRNA based on bipedal 3D DNA walker-driven proximal catalytic hairpin assembly (P-CHA) was developed. P-CHA hairpins were hybridized to a linker DNA strand coupled with magnetic nanoparticles to increase their local concentrations. The bipedal DNA walking on the surface of NPs accelerates reaction kinetics using the proximity effect. Taking advantage of the signal amplification of P-CHA as well as the rapid reaction rate of the DNA walker in 80 min, the proposed sensor detects TK1 mRNA with a low detection limit of 14 pM and may then be applied to clinical diagnosis.

Detection of long mRNA sequences by a Y-shaped DNA probe with three target-binding segments.

Tumor-related mRNA detection is significant and interesting. The current mRNA detection method has the challenge of quantifying long mRNA sequences. Herein, a Y-shaped DNA probe with three target-binding segments was developed to detect tumor-related mRNA. This Y-shaped DNA probe (Y-probe) was assembled by six single DNA strands. Among these DNA strands, two DNA strands contained the split G-quadruplex sequence, and two DNA strands were modified with a pair of fluorophore and quencher, which were used to produce the detectable signal. In the presence of a long target mRNA sequence, target mRNA was hybridized with the three target-binding segments of the Y-probe, resulting in the increased fluorescence of G-quadruplex specific dye Thioflavin T and the decreased fluorescence of fluorophore, which could achieve the ratio detection of target mRNA. The Y-probe exhibited a low detection limit of 17.53 nM. Moreover, this probe showed high accuracy due to the benefits of three target-binding segments.

A novel cationic covalent organic framework as adsorbent for simultaneous removal of methyl orange and hexavalent chromium.

The simultaneous removal of toxic, carcinogenic organic dyes and metal ions from water by one material offers significant advantages when fast, facile, and robust water purification is required. Ionic covalent organic frameworks (ICOFs) have the combined properties of COFs and ion exchange resins and are expected to achieve simultaneous capture of heavy metal ions and organic dyes from water. Herein, a novel guanidinium-based ICOF was synthesized using a solvothermal method. Benefitting from the cationic character, porosity and nanoscale pore size of ICOFs, the adsorbent exhibited high simultaneous adsorption capacities of 290 mg g-1 and 158 mg g-1 for methyl orange (MO) and Cr(vi), respectively, and retained more than 90% adsorption capacity after six adsorption-desorption cycles. In addition, based on dual control of size-exclusion and charge-selection, precisely selective adsorption is achieved towards diverse mixed anionic and cationic pollutants. This strategy offers a practical solution for COFs to confront environmental pollution issues.

Stimulus-responsive strategy based on MnO2 nanosheet-modified mesoporous silica nanoprobes for accurate multiple mRNAs detection.

Fluorescence detection of multiple mRNAs has attracted great attention for disease diagnosis. In this work, a stimulus-responsive strategy for highly sensitive and accurate multiple mRNAs detection was proposed. This stimulus-responsive detection system was prepared by mesoporous silica nanoparticles (MSN), manganese dioxide (MnO2) nanosheets, and DNA probes. DNA probes were loaded into the pores of MSN, which were closed with MnO2 nanosheets. In the presence of glutathione (GSH) and target mRNAs, MnO2 nanosheets were degraded by GSH, resulting in the release of DNA probes. These DNA probes hybridized to the corresponding target mRNA, thereby changing the fluorescence intensity of fluorophores of DNA probes, which could achieve the quantification of target mRNA. This system could simultaneously detect survivin mRNA and Thymidine kinase 1 mRNA at low background levels with relative limits of detection of 0.9 nM and 0.7 nM, respectively. Moreover, this assay has been successfully applied to detect multiple mRNAs with adequate anti-interference ability in the biological sample.

pH-activated DNA nanomachine for miRNA-21 imaging to accurately identify cancer cell.

MicroRNA (miRNA) imaging has been employed to distinguish cancer cells from normal cells by exploiting the overexpression of miRNA in cancer. Inspired by the acidic extracellular tumor microenvironment, we designed a pH-activated DNA nanomachine to enable the specific detection of cancer cells using miRNA imaging. The DNA nanomachine was engineered by assembling two hairpins (Y1 and Y2) onto the surface of a ZIF-8 metal-organic framework (MOF), which decomposed under acidic conditions to release the adsorbed DNA hairpin molecules in situ. The released hairpins were captured by the target miRNA-21 and underwent catalytic hairpin assembly amplification between Y1 and Y2. The detection limit for miRNA assays using the DNA nanomachine was determined to be 27 pM, which is low enough for sensitive detection in living cells. Living cell imaging of miRNA-21 further corroborated the application of the DNA nanomachine in the identification of cancer cell.

A novel heterozygous missense variant of the ARID4A gene identified in Han Chinese families with schizophrenia-diagnosed siblings that interferes with DNA-binding activity.

ARID4A plays an important role in regulating gene expression and cell proliferation. ARID4A belongs to the AT-rich interaction domain (ARID)-containing family, and a PWWP domain immediately precedes its ARID region. The molecular mechanism and structural basis of ARID4A are largely unknown. Whole-exome sequencing (WES) revealed that a novel heterozygous missense variant, ARID4A c.1231 C > G (p.His411Asp), was associated with schizophrenia (SCZ) in this study. We determined the crystal structure of the PWWP-ARID tandem at 2.05 Å, revealing an unexpected mode in which ARID4A assembles with its PWWP and ARID from a structural and functional supramodule. Our results further showed that compared with the wild type, the p.His411Asp ARID mutant protein adopts a less compact conformation and exhibits a weaker dsDNA-binding ability. The p.His411Asp mutation decreased the number of cells that were arrested in the G0-G1 phase and caused more cells to progress to the G2-M phase. In addition, the missense mutation promoted the proliferation of HEK293T cells. In conclusion, our data provide evidence that ARID4A p.His411Asp could cause a conformational change in the ARID4A ARID domain, influence the DNA binding function, and subsequently disturb the cell cycle arrest in the G1 phase. ARID4A is likely a susceptibility gene for SCZ; thus, these findings provide new insight into the role of ARID4A in psychiatric disorders.

An enzyme-free probe based on G-triplex assisted by silver nanocluster pairs for sensitive detection of microRNA-21.

A sensitive ratiometric fluorescence probe based on hybridization chain reaction (HCR) was constructed for sensitive detection of miRNA-21 by using G-triplex and silver nanocluster pairs (AgNC pairs) as an enzyme-free and label-free signal output group. miRNA-21 was used as the primer for the hybridization chain reaction of molecular beacon 1 (MB1) containing the locked G-triplex sequence and molecular beacon 2 (MB2) with intact AgNC pairs at the 5' and 3' end activation. The double-stranded product was obtained along with the opening of the G-triplex and the separation of the AgNC pairs. A detection limit of 67 pM and a linear detection range of 0.1-300 nM were obtained for miRNA-21 determination. The proposed strategy enabled the monitoring of miRNA-21 levels in at least three cell lines, indicating that it provided new ideas for detecting miRNA in real samples. MB1 and MB2 contained the locked G-triplex sequence and silver nanocluster pairs (AgNC pairs), respectively. In the presence of target, the hybridization chain reaction (HCR) between MB1 and MB2 was initiated. At the same time, the locked G-triplex was released and combined to the dye thioflavin T (THT) to increase fluorescence, while the separation of the AgNC pairs caused the fluorescence to decrease. The double-stranded (ds) DNA product was generated to form a ratiometric signal to be detected.

Single-excited double-emission CdTe@CdS quantum dots for use in a fluorometric hybridization assay for multiple tumor-related microRNAs.

A method is described for the simultaneous determination of hepatocellular carcinoma-associated microRNA-122 and microRNA-199a/b-3p. This probe consists of two kinds of nanomaterials. The first comprises CdTe@CdS core-shell quantum dots which, on excitation at 375 nm give two emissions, with peak wavelengths at 543 (g-QDs) and at 627 nm (r-QDs). The second comprises gold nanoparticles acting as a quencher. In the absence of the target, g-QD-N1 and r-QD-N2 are stable due to the fluorescence stability. With the addition of microRNA-122 and microRNA-199a/b-3p, g-QD-N1 and r-QD-N2 are conjugated to the surface of AuNP-S1/S2 through base complementary pairing. As a result, fluorescence resonance energy transfer (FRET) occurs, resulting in a decrease at 550 nm and 635 nm respectively, which can realize the simultaneous detection of two different microRNAs. Detection is achieved within 50 min. The detection limits (3σ/k) are 0.2 nM for microRNA-122 and 0.5 nM for microRNA-199a/b-3p. The clinical applicability of the assay was demonstrated by detecting microRNAs in human serum and different cell lysates. Graphical abstractSchematic for the simultaneous determination of microRNA-122 and microRNA-199a/b-3p by FRET.

A rapid label- and enzyme-free G-quadruplex-based fluorescence strategy for highly-sensitive detection of HIV DNA.

Because rapid, convenient, and selective methods for HIV detection are urgently needed, herein, a simple label-free and enzyme-free strategy is constructed for sensitive fluorescence detection of HIV DNA using the fluorescent intercalating dye thioflavin T (THT) as the detection signal source. This strategy utilizes a hairpin DNA sequence (H1) and two assistant strands. H1 is wisely designed with a G-quadruplex sequence in the stem. Target DNA, when present in solution, will hybridize with H1 to form H1/target duplexes and release the G-quadruplexes. Additionally, the assistant probes hybridize with the unfolded H1 to form a stable DNA double strand, resulting in the displacement of the target to participate in another similar reaction cycle. Consequently, many G-quadruplex structures are generated, leading to a significantly amplified fluorescence signal of THT. The linear range is from 0.1 nM to 50.0 nM with a limit of detection of 13 pM. Results can be achieved within 40 min, because the cyclic amplification involves only one DNA hairpin and two auxiliary chains. Furthermore, this platform exhibited good selectivity with one base mismatch or other DNA sequences. This strategy could be used as a simple, sensitive, and selective tool to detect other DNA biomarkers.

An ultrasensitive guanine wire-based resonance light scattering method using G-quadruplex self-assembly for determination of microRNA-122.

An enzyme-free resonance light scattering (RLS) method is described for the determination of microRNA-122. A guanine nanowire (G-wire) is used that consists of a predesigned DNA1 and a G-quadruplex sequence DNA2. These hybridize with microRNA-122 and partially hybridize with DNA2. After formation of stable double strands with DNA1, DNA2 is released. On addition of K+ and Mg2+ ions, the G-quadruplex sequences undergo self-assembly to form long filamentous G-wires. This increases the intensity of RLS. A 6.1 pM detection limit was obtained, and the linear response covers the 50 pM to 300 nM microRNA concentration range. The method was successfully applied to the quantitation of microRNA-122 in hepatocellular carcinoma cell lysates. Conceivably, this assay can be extended to other RLS methods for biomarker detection by simply changing the sequence of DNA1. Graphical abstract The G-quadruplex sequences of DNA2 were locked with DNA1. The G-quadruplex fragments of DNA2 were released after the hybridization of microRNA-122 with DNA1. These liberated G-quadruplex sequences were self-assembled into long filamentous guanine nanowires (G-wires) which increased resonance light intensity in the presence of Mg2+.

A general scheme for fluorometric detection of multiple oligonucleotides by using RNA-cleaving DNAzymes: application to the determination of microRNA-141 and H5N1 virus DNA.

A widely applicably method is described for fluorometric determination of targets such as microRNA and viral DNA. It is making use of a Mg(II)-dependent DNAzyme and a G-quadruplex. In the absence of analyte, an inactive DNAzyme is formed by the hybridization of split DNAzymes and substrate. On addition of target analyte, the end of each strand of the split DNAzymes bind the analyte. This leads to the generation of an active DNAzyme. In the presence of Mg(II), the activated DNAzyme is formed and can cleave the substrate strand. Hence, the caged G-quadruplex sequences will be released. These released G-quadruplexes combine with thioflavin T to generate a G-quadruplex/thioflavin T complex and thereby cause amplified fluorescence. The method shows a 70 pM detection limit for H5N1 and works over a wide linear range 1 nM to 400 nM. Conceivably, this detection scheme has a wide scope in that it may be applied to other assays for microRNAs and DNAs by variation of the type of DNAzyme. Graphical abstract Schematic presentation of target detection: the DNAzyme cannot cleave the substrate strand when target is absent. Once the target is added, the active DNAzyme can cleave the substrate strand in the presence of Mg2+, resulting in significant fluorescence enhancement when the release of the caged G-quadruplex sequences binding with 2-[4-(dimethylamino)phenyl]-3,6-dimethylbenzothiazolium chloride (ThT).

Screening DNA-targeted anticancer drug in vitro based on cancer cells DNA-templated silver nanoclusters.

A reliable and sensitive cancer cells DNA-templated silver nanoclusters probe has been proposed for screening DNA-targeted anticancer drugs in vitro. In this paper, the DNA-templated silver nanoclusters was used to investigate the binding between anthracycline antibiotics and DNA. The template of DNA-templated silver nanoclusters was extracted from human liver carcinoma cells directly, which can express the drug activities against cancer cells more direct than the normal cells DNA. The anti-tumor activities of the four drugs were validated by MTT and apoptotic assay as Mitoxantrone > Epirubicin > Daunorubicin > Adriamycin.

Double G-quadruplexes in a copper nanoparticle based fluorescent probe for determination of HIV genes.

A DNA-templated copper nanoparticle (CuNP) probe has been developed for the determination of the human immunodeficiency virus oligonucleotide (HIV-DNA). The function of the probe relies on affinity binding-induced DNA hybridization associated with the use of double G-quadruplexes. Double-stranded DNA (dsDNA) with poly(AT-TA) bases was used as a template for synthesis of dsDNA-CuNPs. These have weak fluorescence. In the next step, two G-rich sequences that are linked to both sides of the ds-DNA are locked by HIV complementary DNA (cDNA). If HIV-DNA is introduced, it will hybridize with cDNA, thereby transforming the two G-rich sequences into G-quadruplexes. This enhances the fluorescence of the adjacent dsDNA-CuNPs. Fluorescence increases linearly in the 1 to 200 and 250-1000 nM HIV-DNA concentration range, and the detection limit is 13 pM. This enzyme-free fluorometric assay is time-saving, easily operated, and therefore has large potential in biosensing because it may be extended to various other DNA targets. Graphic abstract Double-strand DNA-templated copper nanoparticles (DNA-CuNPs) have weak fluorescence. When Human Immunodeficiency Virus oligonucleotide (HIV-DNA) is added, it completely hybridized with HIV complementary DNA (cDNA). As a result, the two exposed G-rich sequences are transformed into G-quadruplexes, and an apparent increase in the fluorescence intensity can be observed. (AA: ascorbic acid).

Simply and sensitively simultaneous detection hepatocellular carcinoma markers AFP and miRNA-122 by a label-free resonance light scattering sensor.

In this study, an intelligent and label-free sensor is utilized for the first time to one-spot simultaneous detection hepatocellular carcinoma markers AFP and miRNA-122 by a resonance light scattering (RLS) sensor. cDNA1 hybridizes with cDNA2 to form double-stranded DNA (dsDNA). The construction of dsDNA and methyl violet is used to form the RLS sensor via the electronic interaction. When AFP or miRNA-122 is present, the cDNA (cDNA1 or cDNA2) can bindings of target, thereby RLS intensity changed proportionally with the concentration of AFP or that of miRNA-122. The detection limits of AFP and miRNA-122 are 0.94 µg/L and 98 pM respectively, and their good linear which ranges from 5 to 100 µg/L and 200 pM to 10 nM are achieved using the assay. In the presence of miRNA-122 and AFP mixtures, AFP bound to the AFP aptamer to increase the RLS signal, and miRNA-122 bound to the miRNA-122 complementary strand to decrease the RLS signal. The RLS signal changed in response to changing AFP and miRNA-122 concentrations, so that one-spot simultaneous detection of alpha fetal protein and miRNA-122 is achieved. This method has potential practical applications in the research of hepatocellular carcinoma.

Simple G-quadruplex-based 2-aminopurine fluorescence probe for highly sensitive and amplified detection of microRNA-21.

Based on 2-aminopurine (2-AP) probe in conjunction with a G-quadruplex structure and signal amplification technique, a simple and highly sensitive fluorescence sensor for detecting microRNA (miRNA) is developed for high signal-to-background ratio and wide linear range. The proposed sensor contains two hairpins DNA: H1 and H2. H1 is labeled by 2-AP incorporated into a G-rich sequence. Upon the addition of a target miRNA, H1 is unfolded and forms DNA/RNA complexes that contain a G-quadruplex, thereby significantly enhancing 2-AP fluorescence due to the protection provided by the G-quadruplex. Subsequently, H2 can displace the miRNA from the DNA/RNA complexes and induce signal amplification, resulting in further enhanced fluorescence intensity. Hence, the sensor is highly sensitive and its low limit of detection (L.O.D.) can reach as low as 1.48pM. Furthermore, the proposed sensor is used to detect overexpressed miRNA-21 from human breast cancer cell lysate. The result demonstrates the potential of the proposed sensor to monitor different miRNA biomarkers for the early diagnosis of various cancers.

Ratiometric Fluorescence Sensor for the MicroRNA Determination by Catalyzed Hairpin Assembly.

A novel catalyzed hairpin assembly-based turn-on ratiometric fluorescence biosensor was constructed for the determination of microRNA-122 (miRNA-122) by using 2-aminopurine (2-AP) and thioflavin T (ThT) as detection signal sources. Hairpin DNA sequence (H1) includes the complementary strands of miRNA-122 and G-quadruplex-forming sequence. When miRNA-122 was presented, hybridization occurred between miRNA-122 and part of H1, causing a double-stranded DNA and a G-quadruplex formed. The formed double-stranded DNA significantly decreased the fluorescence intensity of 2-AP. Furthermore, after binding with ThT, the formed G-quadruplex led to the fluorescent enhancement. The hairpin DNA sequence (H2) hybridized with the unfolded H1 and displaced miRNA-122. Finally, the displaced miRNA-122 again hybridized with the H1 and initiated cycle amplification. This sensor showed a linear ranges of 0.5-50 nM and the limit of detection for miRNA-122 assay was 72 pM (with the lowest measured concentration of 500 pM) for determination of miRNA-122 when no other miRNA was present. Measurements on cell lysates from 100, 1000, and 10 000 cells of three different cell lines provided increasing signal ratios, which showed the application potential of the sensor for miRNA determination in real samples.

Simultaneous detection of two hepatocellar carcinoma-related microRNAs using a clever single-labeled fluorescent probe.

The simultaneous detection of two important microRNAs (miRNAs) can potentially evaluate pathological states. The simultaneous detection of miRNA-26a and miRNA-122 is proposed in this study using a cleverly designed single-labeled, dual-functional fluorescent probe with a 2-aminopurine as a fluorophore, which is a G-quadruplex single-stranded DNA. The probe can partly complement cDNA-1 and cDNA-2. cDNA-1 and cDNA-2 are complementary strands of miRNA-26a and miRNA-122, respectively. When the target miRNAs are added simultaneously, these cDNA (cDNA-1 and cDNA-2) can be competed off from the cDNA∖probe duplex to form a cDNA∖RNA heteroduplex. The probe is released and the fluorescent signal is increased. The detection limits of miRNA-26a and miRNA-122 are both 2.5 nM, and their wide linear which ranges from 2.5 to 500 nM are achieved using the assay. This method has potential practical applications.

2-aminopurine probe in combination with catalyzed hairpin assembly signal amplification for simple and sensitive detection of microRNA.

A quencher-free and enzyme-free fluorescent sensor was proposed to simply and sensitively detect miRNA via the target catalyzed hairpin assembly (CHA) signal amplification in combination with 2-aminopurine (2-AP) molecular beacon (MBs). This sensor contains two DNA hairpins termed as H1 and H2. H1 labeled by 2-AP needs no quenchers because 2-AP can be quenched through its stacking interaction with the adjacent bases. H2 is partially complementary to H1. In the presence of the target microRNA (miRNA), H1 is unfolded and produces the DNA/RNA complexes, enhancing the fluorescent signal. Then, the RNA of the DNA/RNA complexes can be displaced by H2 and the free miRNA can interact with another H1, resulting in the significant fluorescence enhancement of the system. This signal amplification process is enzyme-free, making the sensor more simple and cost effective. The detection limit of this sensor could be as low as 3.5pM. We further applied this assay to monitor the overexpressed miRNA-21 from human breast cancer cells to confirm its applicability. The proposed sensor could be used as a simple and sensitive platform for target miRNA detection, holding great potential for convenient monitoring of different miRNA biomarkers for early diagnosis of various cancers.

A novel CdTe quantum dots probe amplified resonance light scattering signals to detect microRNA-122.

We report a rapid and facile resonance light scattering (RLS) technique that utilizes CdTe quantum dots (CdTe QDs) probe to detect microRNA-122. The RLS sensor is ingeniously designed with P1 and P2, two cDNA sequence probes with partially complementary sequences to miRNA-122. The amine-modified P1 and P2 are coupled to the surface of QDs to form functional QDs-P1 and QDs-P2 conjugates, which are collectively referred to as QDs-P. The cDNAs hybridize with the target miRNA to rapidly induce the self-assembly of QDs probe and change RLS intensity. The proposed technique can detect miRNA-122 within 40min. RLS intensity is enhanced in proportion with miRNA-122 concentrations of 0.16-4.80nM and has a low detection limit of 9.4pM. In addition, the assay satisfactorily detects miRNAs in human serum samples. Thus, the assay has considerable potential for the analysis of other interesting tumor makers.

The efficacy assessments of alkylating drugs induced by nano-Fe3O4/CA for curing breast and hepatic cancer.

A new method to evaluate the anticancer activity at the molecular level has been developed. In our assay, the interaction between alkylating anticancer drugs-Fe3O4/CA with DNA has been investigated for the Resonance Light Scattering (RLS) signal enhancement. Water-based nano-Fe3O4, as a probe, has the ability of good solubility, biodegradability and low bulk resistivity etc. The experimental results show that, the activity order of three kinds of drugs is Nimustine (ACNU)>Semustine (Me-CCNU)>Chlormethine (HN2), which is satisfied with the results of the cell apoptosis experiment and the IC50 by MTT method. This assay is simple, sensitive and high efficient. And the theoretical basics for the development of new anticancer drugs as well as the assessments of their efficacy to cure breast and hepatic cancer have been provided.