dsDNA Probe Research Articles

A label-free G-quadruplex-based mercury detection assay employing the exonuclease III-mediated cleavage of T–Hg2+–T mismatched DNA

We report herein the use of an exonuclease III and G-quadruplex probe to construct a G-quadruplex-based luminescence detection platform for Hg2+. Unlike common DNA-based Hg2+ detection methods, when using the dsDNA probe to monitor the hairpin formation, the intercalation of the dsDNA probe may be influenced by the distortion of dsDNA. This ‘mix-and-detect’ methodology utilized the G-quadruplex probe as the signal transducer and is simple, rapid, convenient to use and can detect down to 20 nM of Hg2+.

A dual amplification fluorescent strategy for sensitive detection of DNA methyltransferase activity based on strand displacement amplification and DNAzyme amplification.

DNA methyltransferase (MTase) plays a critical role in many biological processes and has been regarded as a predictive cancer biomarker and a therapeutic target in cancer treatment. Sensitive detection of DNA MTase activity is essential for early cancer diagnosis and therapeutics. Here, we developed a dual amplification fluorescent strategy for sensitive detection of DNA MTase activity based on strand displacement amplification (SDA) and DNAzyme amplification. A trifunctional double-stranded DNA (dsDNA) probe was designed including a methylation site for DNA MTase recognition, a complementary sequence of 8-17 DNAzyme for synthesizing DNAzyme, and a nicking site for nicking enzyme cleavage. Firstly, the trifunctional dsDNA probe was methylated by DNA MTase to form the methylated dsDNA. Subsequently, HpaII restriction endonuclease specifically cleaved the residue of unmethylated dsDNA. Next, under the action of polymerase and nicking enzyme, the methylared dsDNA initiated SDA, releasing numbers of 8-17 DNAzymes. Finally, the released 8-17 DNAzymes triggered DNAzyme amplification reaction to induce a significant fluorescence enhancement. This strategy could detect DNA MTase activity as low as 0.0082U/mL. Additionally, the strategy was successfully applied for evaluating the inhibitions of DNA MTase using two anticancer drugs, 5-azacytidine and 5-aza-2'-deoxycytidine. The results indicate the proposed strategy has a potential application in early cancer diagnosis and therapeutics.

Fok I cleavage–inhibition strategy for the specific and accurate detection of transcription factors

Specific and accurate detection of transcription factors is critical for disease diagnosis and drug development. Here, we developed a novel and versatile fluorescent sensing strategy for specific and accurate detection of transcription factors based on the inhibition of endonuclease Fok I-catalyzed DNA cleavage reaction. A FAM-labeled double-stranded DNA probe (dsDNA probe) with transcription factor binding site and Fok I recognition site was designed for target recognition and signal transduction. With the binding of transcription factors, the dsDNA probes are protected from cleavage by Fok I. These protected dsDNA probes then cannot hybridize with the added BHQ-DNA, keeping the fluorescence of FAM in an on state. However, in the absence of targets, the dsDNA probes were cleaved to release the FAM-DNA, which subsequently hybridized with BHQ-DNA, resulting in the fluorescence of the FAM being quenched. With the Fok I–DNA interaction that can specifically recognize the transcription factor-DNA binding and accurately convert the detection of transcription factors to the detection of DNA, the proposed strategy realized the reliable detection of model target NF-κB p50 with a nanomolar detection limit. This strategy was also employed to detect the inhibition effect of oridonin, a known inhibitor of NF-κB. Furthermore, perfect recoveries were obtained when detecting the targets in HeLa cells lysate, demonstrating the feasibility of this strategy for transcription factor detection in biological samples.

Simple and signal-off electrochemical biosensor for mercury(II) based on thymine-mercury-thymine hybridization directly on graphene

A simple, signal-off and reusable electrochemical biosensor was developed for sensitive and selective detection of mercury(II) based on thymine–mercury(II)–thymine (T–Hg2+–T) complex and the remarkable difference in the affinity of graphene with double strand DNA (ds-DNA) and single strand DNA (ss-DNA). Our system was composed of ferrocene-tagged probe DNA and graphene. Due to the noncovalent assembly, the ferrocene-tagged probe ss-DNA was immobilized on the surface of graphene nanosheets directly and employed to amplify the electrochemical signal. In the presence of Hg2+, the ferrocene-labeled T-rich DNA probe hybridized with target probe to form ds-DNA via the Hg2+-mediated coordination of T-Hg2+-T base pairs. As a result, the duplex DNA complex kept away from the graphene surface due to the weak affinity of graphene and ds-DNA, and the redox current decreased substantially. Meanwhile, the graphene decorated GCE surface was released for the reusability. Under the optimal conditions, the proposed sensor showed a linear concentration range from 25pM to 10μM with a detection limit of 5pM for Hg2+ detection. The strategy afforded exquisite selectivity for Hg2+ against other metal ions in real environmental samples.

Dual-probe electrochemical DNA biosensor based on the "Y" junction structure and restriction endonuclease assisted cyclic enzymatic amplification for detection of double-strand DNA of PML/RARα related fusion gene.

Taking advantage of "Y" junction structure and restriction endonuclease assisted cyclic enzymatic amplification, a dual-probe electrochemical DNA (DE-DNA) biosensor was designed to detect double-stranded DNA (dsDNA) of acute promyelocytic leukemia (APL) related gene. Two groups of detection probes were designed, and each group was composed of a biotinylated capture probe and an assisted probe. They were separately complementary with two strands of target dsDNA in order to prevent the reannealing of the two separate strands from target dsDNA. First, thiol functionalized capture probes (C1 and C2) were severally assembled onto two different gold electrodes, followed by hybridizing with target dsDNA (S1a-S1b) and assistant probes to form two Y-junction-structure ternary complexes. Subsequently, restriction sites on the ternary complexes were digested by Rsa I, which can release S1a, S1b and biotins from the electrode surfaces. Meanwhile, the released S1a and S1b can further hybridize with the unhybridized corresponding detection probes and then initiate another new hybridization-cleavage-separation cycle. Finally, the current signals were produced by the enzyme-catalyzed reaction of streptavidin-horse reddish peroxidase (streptavidin-HRP). The distinct difference in current signals between different sequences allowed detection of target dsDNA down to a low detection limit of 47 fM and presented excellent specificity with discriminating only a single-base mismatched dsDNA sequence. Moreover, this biosensor was also used for assay of polymerase chain reaction (PCR) samples with satisfactory results. According to the results, the power of the DE-DNA biosensor as a promising tool for the detection of APL and other diseases.

DNA-binding studies of complex of Pt(bpy)(pip)]2+ and [Pt(bpy)(hpip)]2+ by electrochemical methods: development of an electrochemical DNA biosensor

The electrochemical properties and deoxyribonucleic acid (DNA)-binding affinities of [Pt(bpy)(pip)]2+ (1) and [Pt(bpy)(hpip)]2+ (2) in homogeneous solution were studied. These platinum(II) complexes have shown to interact with DNA via intercalation mode. For the cyclic voltammetry (CV) experiments, since 2 leads to a larger decrease in the peak current and more positive shift in the peak potential in comparison to the analogous compound of 1, it could be noted that 2 exhibits higher intercalative binding affinity against DNA. The effect of ionic strength and competitive binding studies in the presence of ethidium bromide (EB) were also investigated by CV. Electrochemical DNA biosensor based on the immobilization of double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) probe and 1 and 2 onto electrochemically activated glassy carbon (GC) electrode was also achieved. The immobilization of dsDNA and ssDNA probe and hybridization were monitored by differential pulse voltammetry using 1 and 2 as hybridization indicators. It was found that both platinum complexes show larger and more evident electrochemical signals for the hybridized probe dsDNA with respect to ssDNA immobilized on GC electrode. The developed electrochemical DNA biosensor showed good selectivity and analytical performance for the complementary target nucleotide with limit of detection of 1.23 × 10−8 and 8.05 × 10−9 mol L−1 and limit of quantification of 4.06 × 10−7 and 2.66 × 10−8 for 1 and 2, respectively.

A DNA machine-based fluorescence amplification strategy for sensitive detection of uracil-DNA glycosylase activity

Sensitive detection of uracil-DNA glycosylase (UDG) activity is critical for function study of UDG and clinical diagnosis. Here, we developed a novel fluorescent strategy for sensitive detection of UDG activity based on the signal amplification by a label-free and enzyme-free DNA machine. A double-strand DNA (dsDNA) probe P1–P2 with uracil bases and trigger sequence was designed for UDG recognition and signal transduction. Two hairpin probes H1 and H2 which were partially complementary were employed to construct the label-free and enzyme-free DNA machine. Under the action of UDG, uracil bases were removed from the P1-P2 dsDNA probe, and then a strand P2ʹ with abasic sites was released. Subsequently, the liberated P2ʹ activated the DNA machine and generated numerous H1–H2 complexes containing G-quadruplex (G4) structures in the end. Finally, the G4 structures could bind with N-methylmesoporphyrin IX (NMM) to form G4-NMM complexes with the enhanced fluorescence responses. This strategy could detect UDG activity as low as 0.00044U/mL. In addition, the strategy was also applied for the analysis of UDG activity in HeLa cells lysate with low effect of cellular components. Moreover, this strategy was successfully applied for assaying the inhibition of UDG using uracil glycosylase inhibitor (UGI). This strategy provided a potential tool for sensitive quantification of UDG activity in UDG functional study and clinical diagnosis.

Target-induced reconfiguration of DNA probes for recycling amplification and signal-on electrochemical detection of hereditary tyrosinemia type I gene.

By coupling target DNA-induced reconfiguration of the dsDNA probes with enzyme-assisted target recycling amplification, we describe the development of a signal-on electrochemical sensing approach for sensitive detection of hereditary tyrosinemia type I gene. The dsDNA probes are self-assembled on the sensing electrode, and the addition of the target DNA reconfigures and switches the dsDNA probes into active substrates for exonuclease III, which catalytically digests the probes and leads to cyclic reuse of the target DNA. The target DNA recycling and the removal of one of the ssDNA from the dsDNA probes by exonuclease III result in the formation of many hairpin structures on the sensor surface, which brings the electroactive methylene blue labels into proximity with the electrode and produces a significantly amplified current response for sensitive detection of the target gene down to 0.24 pM. This method is also selective to discriminate single-base mismatch and can be employed to detect the target gene in human serum samples. With the demonstration for the detection of the target gene, we expect the developed method to be a universal sensitive sensing platform for the detection of different nucleic acid sequences.

An electrochemical method to evaluate p53 C-terminal domain acetylation on its DNA binding ability

In this work, an electrochemical method has been developed to evaluate the DNA binding efficiency of p53 C-terminal domain (CTD) after acetylation. Firstly, peptides derived from the CTD of p53 with single or multiple acetylated lysine residues are immobilized on an electrode surface. Then, ferrocene (Fc) labeled dsDNA probe bind with the p53 CTD and give different electrochemical response corresponding to the acetylation degree. Consequently, the electrochemical signals can clearly distinguish a different extent of p53 CTD acetylation on its DNA binding ability. Experimental results also show that acetylation of p53 CTD may enhance its DNA binding ability which is positively correlated with the acetylated lysine residue numbers. This electrochemical method is simple, which may provide a novel method to study protein modifications in the future.

An Host-Guest Electrochemical Sensor for DNA Ligase Detection

We herein constructed a electrochemial sensor for T4 DNA ligase detction. In the sensor, the hairpin dsDNA terminal-labeled with 4-((4-(dimethylamino)phenyl)azo)benzoic acid (dabcyl) and thiol group was immobilized on Au electrode surface as the probe by Au-S bond, and the CdS nanoparticles surface-modified with β-cyclodextrins (ZnS-CD) were employed as electrochemical signal provider and host-guest recognition element. After T4 DNA ligase repairing single-strand breaks in duplex DNA, the dsDNA probe underwent a significant conformational change, which forced dabcyl away from the electrode. As a result, formerly shielded dabcyl became accessible to host-guest recognition between β-cyclodextrin and dabcyl, thus the target repairing event could be sensitively transduced to electrochemical signal provided by ZnS-CD.

Multiplexed electrochemical coding of DNA–protein bindings

A simple, sensitive and multiplexed electrochemical sensor for the detection of DNA–protein binding based on the exonuclease protection strategy is described. Two electroactive species, methylene blue (MB)- and ferrocene (Fc)-labeled dsDNA probes are self-assembled on a gold electrode to prepare the sensor surface. The target proteins, vascular endothelial growth factor (VEGF) and estrogen receptor (ERα), bind to the dsDNA probes and protect the probes from digesting by exonuclease III due to the steric hindrance of the bound proteins. These protein-protected, MB/Fc-labeled sequences remaining on the sensor surface display two distinct voltammetric peaks, whose peak potentials (MB: −0.27V; Fc: +0.27V) and intensities reflect the identities and amounts of the corresponding target proteins, for simultaneous and multiplexed detection of DNA–protein bindings. The proposed sensor is also selective to the target proteins against other interference molecules. By using labels with distinct voltammetric peaks, the developed method can be easily expanded for simultaneous detection of multiple DNA–protein bindings.

Toehold strand displacement-driven assembly of G-quadruplex DNA for enzyme-free and non-label sensitive fluorescent detection of thrombin

Based on a new signal amplification strategy by the toehold strand displacement-driven cyclic assembly of G-quadruplex DNA, the development of an enzyme-free and non-label aptamer sensing approach for sensitive fluorescent detection of thrombin is described. The target thrombin associates with the corresponding aptamer of the partial dsDNA probes and liberates single stranded initiation sequences, which trigger the toehold strand displacement assembly of two G-quadruplex containing hairpin DNAs. This toehold strand displacement reaction leads to the cyclic reuse of the initiation sequences and the production of DNA assemblies with numerous G-quadruplex structures. The fluorescent dye, N-Methyl mesoporphyrin IX, binds to these G-quadruplex structures and generates significantly amplified fluorescent signals to achieve highly sensitive detection of thrombin down to 5pM. Besides, this method shows high selectivity towards the target thrombin against other control proteins. The developed thrombin sensing method herein avoids the modification of the probes and the involvement of any enzyme or nanomaterial labels for signal amplification. With the successful demonstration for thrombin detection, our approach can be easily adopted to monitor other target molecules in a simple, low-cost, sensitive and selective way by choosing appropriate aptamer/ligand pairs.

A label-free fluorescence strategy for sensitive detection of ATP based on the ligation-triggered super-sandwich

In this study, a label-free fluorescence strategy for sensitive detection of ATP based on the ligation-triggered super-sandwich is reported. We designed a double-stranded DNA (ds-DNA) probe as the substrate of ATP-dependent ligation. SYBR Green I (SG I), a double-duplex DNA specific dye, was employed as the readout signal. In the absence of ATP, the ligation would not occur and the ds-DNA remained intact. Further, a weak fluorescence could be observed due to the intercalation of SG I into the grooves of the ds-DNA probe. In the presence of ATP, T4 DNA ligase would catalyse the ligation between 3ʹ-OH and 5ʹ-PO4 ends between ds-DNA probes. As a result, more binding sites of the SG I were generated and a fluorescence enhancement was obtained. This method showed a good sensitivity with a detection limit of 200pM and could perfectly discriminate ATP from its analogs.

An New Electrochemical Sensor for DNA Ligase Detection

We herein constructed a sensor that converts T4 DNA ligase-induced conformational transformation of the probe DNA to electrochemical response based on host-guest recognition and nanoparticle label. In the sensor, the hairpin dsDNA terminal-labeled with 4-((4-(dimethylamino)phenyl)azo)benzoic acid (dabcyl) and thiol group was immobilized on Au electrode surface as the probe by Au-S bond, and the CdS nanoparticles surface-modified with β-cyclodextrins (CdS-CD) were employed as electrochemical signal provider and host-guest recognition element. After T4 DNA ligase repairing single-strand breaks in duplex DNA, the dsDNA probe underwent a significant conformational change, which forced dabcyl away from the electrode. As a result, formerly shielded dabcyl became accessible to host-guest recognition between β-cyclodextrin and dabcyl, thus the target repairing event could be sensitively transduced to electrochemical signal provided by CdS-CD.

Target recycling amplification for label-free and sensitive colorimetric detection of adenosine triphosphate based on un-modified aptamers and DNAzymes

Based on target recycling amplification, the development of a new label-free, simple and sensitive colorimetric detection method for ATP by using un-modified aptamers and DNAzymes is described. The association of the model target molecules (ATP) with the corresponding aptamers of the dsDNA probes leads to the release of the G-quadruplex sequences. The ATP-bound aptamers can be further degraded by Exonuclease III to release ATP, which can again bind the aptamers of the dsDNA probes to initiate the target recycling amplification process. Due to this target recycling amplification, the amount of the released G-quadruplex sequences is significantly enhanced. Subsequently, these G-quadruplex sequences bind hemin to form numerous peroxidase mimicking DNAzymes, which cause substantially intensified color change of the probe solution for highly sensitive colorimetric detection of ATP down to the sub-nanomolar (0.33nM) level. Our method is highly selective toward ATP against other control molecules and can be performed in one single homogeneous solution, which makes our sensing approach hold great potential for sensitive colorimetric detection of other small molecules and proteins.

A label-free electrochemical DNA sensor using methylene blue as redox indicator based on an exonuclease III-aided target recycling strategy

In this work, using methylene blue (MB) as a redox marker and exonuclease III (Exo III) as an amplificatory enzyme, we developed a facile and a label-free electrochemical method for sensitive DNA detection. A double-stranded DNA (dsDNA) probe was prepared by hybridizing two single-stranded DNA (ssDNA) probes. In the ssDNA probes, one ssDNA was guanine bases free and the other one consisted of many unbound guanine bases. MB could be absorbed on the unbound guanine bases owing to the specific interaction between MB and the guanine bases. When the dsDNA probe was challenged with target DNA, it induced a simple Exo III assisted cleavage process, accompanied by the release of the unbound guanine bases. Thus, the amount of MB absorbed on the electrode was much less compared to the initial signal. The detection limit for DNA was found to be as low as 20fM. Moreover, it could discriminate mismatched DNA from perfectly matched target DNA. This detection method is simple in design, fast in operation and can be applied to detect different DNA sequences.

Screening for Protein-DNA Interactions by Automatable DNA-Protein Interaction ELISA

DNA-binding proteins (DBPs), such as transcription factors, constitute about 10% of the protein-coding genes in eukaryotic genomes and play pivotal roles in the regulation of chromatin structure and gene expression by binding to short stretches of DNA. Despite their number and importance, only for a minor portion of DBPs the binding sequence had been disclosed. Methods that allow the de novo identification of DNA-binding motifs of known DBPs, such as protein binding microarray technology or SELEX, are not yet suited for high-throughput and automation. To close this gap, we report an automatable DNA-protein-interaction (DPI)-ELISA screen of an optimized double-stranded DNA (dsDNA) probe library that allows the high-throughput identification of hexanucleotide DNA-binding motifs. In contrast to other methods, this DPI-ELISA screen can be performed manually or with standard laboratory automation. Furthermore, output evaluation does not require extensive computational analysis to derive a binding consensus. We could show that the DPI-ELISA screen disclosed the full spectrum of binding preferences for a given DBP. As an example, AtWRKY11 was used to demonstrate that the automated DPI-ELISA screen revealed the entire range of in vitro binding preferences. In addition, protein extracts of AtbZIP63 and the DNA-binding domain of AtWRKY33 were analyzed, which led to a refinement of their known DNA-binding consensi. Finally, we performed a DPI-ELISA screen to disclose the DNA-binding consensus of a yet uncharacterized putative DBP, AtTIFY1. A palindromic TGATCA-consensus was uncovered and we could show that the GATC-core is compulsory for AtTIFY1 binding. This specific interaction between AtTIFY1 and its DNA-binding motif was confirmed by in vivo plant one-hybrid assays in protoplasts. Thus, the value and applicability of the DPI-ELISA screen for de novo binding site identification of DBPs, also under automatized conditions, is a promising approach for a deeper understanding of gene regulation in any organism of choice.

Label-free electrochemical biosensor using home-made 10-methyl-3-nitro-acridone as indicator for picomolar detection of nuclear factor kappa B

A new acridone derivative 10-methyl-3-nitro-acridone (MNA) with excellent electrochemical activity was synthesized in this paper. Using it as an electrochemical indicator, a signal-on and label-free electrochemical biosensor was developed for picomolar determination of nuclear factor kappa B (NF-κB) in serum. Initially, linear double-stranded DNA (dsDNA) probes, which contain a protein-binding site specific to NF-κB, were self-assembled on the surface of a glass carbon electrode (GCE). If the NF-κB was absent, the dsDNA probes were cut into ss-DNA fragments by the digestion of ExoIII, resulting in a low electrochemical signal of MNA due to the weak binding affinity of MNA to ss-DNA. On the contrary, in the presence of NF-κB, it could bind with the dsDNA probes at the specific site and hinder the digestion of ExoIII, resulting in a significant increase of electrochemical response due to the intercalation of MNA into the dsDNA probes. By employing the above strategy, this sensor could detect as low as 40pM NF-κB with high specificity. To the best of our knowledge, the proposed sensor is the first attempt to use acridone derivative as an electrochemical indicator for NF-κB detection, which may represent a promising path toward clinical diagnosis and drug developments.

Design of multiplex logic gates: Combining regulation of DNA structure with logical calculation

In this paper, we proposed a facile and accurate way for controlling multiplex fluorescent logic gates through changing the exciting and the observing wavelengths. As proof-of-principle, a Pb2+-specific DNAzyme probe and a thymine (T)-rich DNA probe were introduced to a double-stranded (ds-) DNA. The addition style of the two ions served as the four inputs by changing the distance of the three fluorophores, 6-carboxyfluorescein (FAM), ALEXA 532 (ALEXA) and carboxytetramethylrhodamine (TAMRA), all of which were modified on the dsDNA probe. Compared with the previous methods, the present approach needed neither different inputs nor the change of sequence of the probe to achieve multiplex logic gates. Furthermore, the modularity of the strategy may allow it to be extended to other types of logic gates.

Sensitive DNA biosensor improved by 1,10-phenanthroline cobalt complex as indicator based on the electrode modified by gold nanoparticles and graphene

A novel and sensitive electrochemical DNA biosensor was constructed based on the gold electrode modified by graphene and gold nanoparticles (AuNPs), whose sensitivity was improved by using 1,10-phenanthroline cobalt ([Co(phen)2(Cl)(H2O)]+) as an electroactive indicator. The gold electrode was firstly modified with sulfur graphene (GR-SH), and then covalently grafted with AuNPs via sulfur–gold affinity. Finally the thiol capped probe DNA was immobilized onto the AuNPs/GR modified electrode. The target DNA could hybridize with the probe DNA to form a double-stranded DNA (ds-DNA), into which [Co(phen)2(Cl)(H2O)]+ can intercalate, which can be confirmed by cyclic voltammetry (CV). The target DNA, from Escherichia coli (E. coil), could be detected in a linear range from 2.50 × 10−11 to 1.25 × 10−9 M (R = 0.9981) with a detection limit of 8.33 × 10−12 M (3σ, n = 11). It was demonstrated that the reported electrochemical DNA biosensor offered a promising approach for the sensitive and selective detection of DNA by using [Co(phen)2(Cl)(H2O)]+ as an electroactive indicator.