Linear DNA Probes Research Articles

DNA-Based Nanomaterials for Analysis of Extracellular Vesicles.

Extracellular vesicles (EVs) are cell-derived nanovesicles comprising a myriad of molecular cargo such as proteins and nucleic acids, playing essential roles in intercellular communication and physiological and pathological processes. EVs have received substantial attention as noninvasive biomarkers for disease diagnosis and prognosis. Owing to their ability to recognize protein and nucleic acid targets, DNA-based nanomaterials with excellent programmability and modifiability provide a promising tool for the sensitive and accurate detection of molecular cargo carried by EVs. In this perspective, recent advancements in EV analysis using a variety of DNA-based nanomaterials are summarized, which can be broadly classified into three categories: linear DNA probes, DNA nanostructures, and hybrid DNA nanomaterials. The design, construction, advantages, and disadvantages of different types of DNA nanomaterials, as well as their performance for detecting EVs are reviewed. The challenges and opportunities in the field of EV analysis by DNA nanomaterials are also discussed.

DNA Nanotechnology-Empowered Fluorescence Imaging of APE1 Activity

Apurinic/apyrimidinic endonuclease 1 (APE1), also known as redox factor-1 (Ref-1), is a multifunctional protein that exists widely in living organisms. It can specifically recognize and cleave the DNA in apurinic/apyrimidinic (AP) sites in the base excision repair (BER) pathway, as well as regulate the expression of genes to activate some transcription factors. The abnormal expression and disruptions in the biological functions of APE1 are linked to a number of diseases, including inflammation, immunodeficiency, and cancer. Hence, it is extremely desired to monitor the activity of APE1, acquiring a thorough understanding of the healing process of damaged DNA and making clinical diagnoses. Thanks to the advent of DNA nanotechnology, some nanodevices are used to image the activity of APE1 with great sensitivity and simplicity. In this review, we will summarize developments in DNA-nanotechnology-empowered fluorescence imaging in recent years for APE1 activity according to different types of DNA probes, which are classified into linear DNA probes, composite DNA nanomaterials, and three-dimensional (3D) DNA nanostructures. We also highlight the future research directions in the field of APE1 activity imaging.

A dual DNA tetrahedrons and MnO2 nanosheets sustained entropy-driven DNA amplifier enables high-performance operation in live cells and bodies under a light-gated manner

Benefiting from the low chance of circuit leakage and the sufficient driving forces, the entropy-driven DNA amplifier has attracted great attention in constructing new fluorescent biosensors to operate in live specimen. But unfortunately, this promising signal amplification means still confront with some inevitable defects such as poor accuracy, inferior bio-stability and unsatisfactory biological uptake efficiency. To deal with these challenges, we propose here a series of rewarding sensing designs. First, an exceptional light-gated manner is integrated by embedding the sensing elements with a photocleavage-linker, under which the target specific recognition is capable of being initiated in a faultless control mode under a light irradiation. Then, dual DNA tetrahedrons are employed to anchor the linear DNA probes on their endpoints to significantly improve the enzyme resistance in complex biological medium. After that, a two-dimensional nanomaterial (MnO2 nanosheets) that can be efficiently reduced by biothiols is selected as an auxiliary nanovector to promote the endocytosis. By virtue of these efforts, this special biosensor is endowed with a good assay performance towards an underlying lung cancer related biomarker (microRNA-196a), wherein the sensitivity is as low as 3.2 pM and the specificity owns an identification ability for single-base mismatching. More importantly, our method can be utilized as a high-performance imaging toolbox for reliably detecting the target in live cancer/normal cells and bodies, offering a broad application prospect for cancer diagnosis.

Long-Term Functional Stability of Functional Nucleic Acid–Gold Nanoparticle Conjugates with Different Secondary Structures

Thiolated functional nucleic acid-gold nanoparticle conjugates (FNA-AuNPs) are the core recognition elements in biosensors. The long-term functional stability (LTFS) is critical for their practical applications and, however, has been overlooked. Here we report on the huge effects of multiple experimental factors on LTFS, including spacer- and buffer-composition, secondary structures of FNAs, and surface blocking. We quantitatively determined these effects by measuring the relative hybridization capacity (RHC, the relative amount of complementary DNA hybridized with the same amount of conjugates) for linear DNA-AuNP or the relative signal change generated by their function (RSC-F) for molecular beacon (MB) and G-quadruplex (G4)-AuNPs. There is a positive relationship between the spacer affinity [oligoadenine (A10) > oligothymine (T10) > oligoethlyene glycol (EG18)] of the linear DNA probes and the LTFS. The LTFS of linear DNA-AuNP in phosphate buffer (PB) was much better than that in Good's buffers such as HEPES, Tris, and MES. The secondary structure of FNAs also strongly impacted the LTFS, showing the substantially decreased LTFS from G4- to linear DNA- to MB-AuNPs, where EG18 spacer was used for all these conjugates. The surface blocking of FNA-AuNPs greatly improved the LTFS. We experimentally determined that the LTFS of FNA-AuNPs was directly related to the dissociation of DNAs caused by the in situ generated H2O2 due to the oxidase activity of AuNP and thereby oxidation of Au-thiol bonds. The oxidase activity of AuNP was favored at high temperature, low pH, high AuNP concentration, high Good's buffer concentration, and high salt concentration, corresponding well with the positive effects of high affinity spacer, PB, and surface blocking on the LTFS of FNA-AuNPs. Our study has implications on both fundamental surface science and practical applications.

NMR solution structure of an asymmetric intermolecular leaped V-shape G-quadruplex: selective recognition of the d(G2NG3NG4) sequence motif by a short linear G-rich DNA probe.

Aside from classical loops among G-quadruplexes, the unique leaped V-shape scaffold spans over three G-tetrads, without any intervening residues. This scaffold enables a sharp reversal of two adjacent strand directions and simultaneously participates in forming the G-tetrad core. These features make this scaffold itself distinctive and thus an essentially more accessible target. As an alternative to the conventional antisense method using a complementary chain, forming an intermolecular G-quadruplex from two different oligomers, in which the longer one as the target is captured by a short G-rich fragment, could be helpful for recognizing G-rich sequences and structural motifs. However, such an intermolecular leaped V-shape G-quadruplex consisting of DNA oligomers of quite different lengths has not been evaluated. Here, we present the first nuclear magnetic resonance (NMR) study of an asymmetric intermolecular leaped V-shape G-quadruplex assembled between an Oxytricha nova telomeric sequence d(G2T4G4T4G4) and a single G-tract fragment d(TG4A). Furthermore, we explored the selectivity of this short fragment as a potential probe, examined the kinetic discrimination for probing a specific mutant, and proposed the key sequence motif d(G2NG3NG4) essential for building the leaped V-shape G-quadruplexes.

Hybridization-initiated exonuclease resistance strategy for simultaneous detection of multiple microRNAs

Multiple miRNA detection is often limited by sample, time-consuming, and complicated procedures. To address such challenges, we present a relatively simple and amplification-free fluorescent strategy based on hybridization-initiated exonuclease resistance for simultaneous detection of multiple miRNAs in a single tube. Single-stranded linear DNA probes were designed with dual roles of capture and reporter DNA, i.e. each DNA probe was labeled with biotin at the 3′-end for signal readout and amino at the 5′-end for probe immobilization. After target miRNAs specifically hybridized with the corresponding probes, the formation of double-stranded probe-target duplex protected the biotin on the probe from Exonuclease I digestion, thus resulting in high fluorescent intensities through the reaction between biotin and streptavidin-phycoerythrin. Coupling with the 3-plex fluorescent microsphere-based assay system, herein we successfully demonstrated the simultaneous and quantitative measurement of three sequence-specific miRNAs at concentration range of 2.5 pM to 1.25 nM and detection limits of 2 pM. To meet high throughput and rapid turnaround time requirements, we have also exemplified that, even shortening the total reaction time within 1 h, wider linear response range and lower detection limit were guaranteed. We further applied this assay to detect endogenous target miRNA levels from five kinds of cancer cell line and one normal cell line HEK 293T. This simple and rapid strategy will hold great potential for monitoring of multiple miRNAs biomarkers in biomedical research and early clinical diagnosis.

Conformational Changes of Single-Stranded Hairpin DNA Covalently Immobilized on Solid Surfaces Investigated by Molecular Dynamics Simulations

Molecular beacons (MBs) are stem-loop hairpin structured nucleic acid probes that have shown greater sensitivity and specificity over linear DNA probes. For high-throughput DNA detection, MBs are often covalently immobilized on a variety of solid surfaces (e.g., glass slides and gold surfaces). The performance of immobilized MBs on solid surfaces has been found to be dependent on the stability and orientation of their stem-loop structures on surfaces. At present, however, there is still a lack of high-resolution experimental characterization approaches for determining the conformational behaviors of immobilized MBs on surfaces. Herein, we investigated the structure and dynamics of immobilized MB probe in NaCl and MgCl2 solutions respectively by performing all-atom molecular dynamics simulations. We found that the single immobilized MB probe maintained a stable stemloop structure in the MgCl2 solution, while it undergone significant structural distortion in the NaCl solution. The results show that Mg2+ ions have a stronger stabilizing effect on immobilized hairpins compared with Na+ ions, which is attributed to their greater shielding ability for the negatively charged phosphate backbone. Moreover, the hydrogen-bonding dynamics in the hairpin's loop and stem parts were analyzed respectively to further understand the effect of the base–base interactions on conformational stability of MBs immobilized on solid surfaces. The findings in this work help to optimize the design of MBs biosensors, thus further improving their performance of nucleic acid detection.

Photoinduced Electron Transfer-Based Fluorescence Quenching Combined with Rolling Circle Amplification for Sensitive Detection of MicroRNA

MicroRNAs (miRNAs) detection is crucial for further understanding the biological functions of miRNAs and early cancer diagnosis and therapeutics, but now remains a great challenge. Here, we present a novel homogenous biosensing strategy for fluorescence detection of miRNA. In this strategy, a preliminary synthesized circular DNA was used as recognition probe for hybridization with miRNA target, and then miRNA target primed a rolling circle amplification (RCA) reaction. The RCA product could be hybridized with thousands of carboxyfluorescein (FAM)-labeled linear DNA probes, which led FAM to be close to -GGG- base of RCA product, accompanying with the significant fluorescence quenching due to photoinduced electron transfer (PET) between FAM and guanine. This is for the first time to integrate RCA and PET into one detection process. With highly efficient amplification of RCA and excellent signal readout of PET, this method exhibited a high sensitivity toward target miRNA with a detection limit of 6 aM. The target-dependent circularization of the padlock probe and the ligation reaction could improve the specificity effectively, leading to discrimination between miRNA family members. This method provides a simple, isothermal, and low-cost approach for sensitive detection of miRNA and holds great potential for early diagnosis in gene-related diseases.

A novel real-time reverse transcription-polymerase chain reaction assay with partially double-stranded linear DNA probe for sensitive detection of hepatitis C viral RNA

The detection and quantification of HCV RNA is very helpful for the management and treatment of HCV related diseases. Detection of low HCV viral load is a great challenge in HCV RNA detection. Here, we developed a novel real-time RT-PCR assay with partially double-stranded linear DNA probe which can detect all HCV genotypes and improve the detection performance. The novel assay has a wide linear dynamic range of HCV RNA quantification (1×102–1×1011IU/ml) and a limit of detection of 78IU/ml. The assay exhibits an excellent reproducibility with 2.52% and 1.33% coefficients of variations, for inter- and intra-assays, respectively. To evaluate the viability of the assay, a comparison with a commercial HCV RNA detection kit was performed using 106 serum samples. The lineared correlation coefficient between the novel assay and the commercial HCV RNA detection kit was 0.940. Meanwhile, the deviation between the two methods was tolerable. Therefore, the novel real-time RT-PCR assay was applicable for laboratory diagnosis and monitoring of HCV infection.

Molecular Crowding Effects on Microgel-Tethered Oligonucleotide Probes.

Microgel tethering is a nontraditional method with which to bind oligonucleotide hybridization probes to a solid surface. Microgel-tethering physically positions the probes away from the underlying hard substrate and maintains them in a highly waterlike environment. This paper addresses the question of whether molecular crowding affects the performance of microgel-tethered molecular beacon probes. The density of probe-tethering sites is controlled experimentally using thin-film blends of biotin-terminated [PEG-B] and hydroxyl-terminated [PEG-OH] poly(ethylene glycol) from which microgels are synthesized and patterned by electron beam lithography. Fluorescence measurements indicate that the number of streptavidins, linear DNA probes, hairpin probes, and molecular beacon probes bound to the microgels increases linearly with increasing PEG-B/PEG-OH ratio. For a given tethering-site concentration, more linear probes can bind than structured probes. Crowding effects emerge during the hybridization of microgel-tethered molecular beacons but not during the hybridization of linear probes, as the tethering density increases. Crowding during hybridization is associated with conformational constraints imposed by the close proximity of closed and hybridized structured probes. The signal-to-background ratio (SBR) of hybridized beacons is highest and roughly constant for low tethering densities and decreases at the highest tethering densities. Despite differences between microgel tethering and traditional oligonucleotide surface-immobilization approaches, these results show that crowding defines an optimum tethering density for molecular beacon probes that is less than the maximum possible, which is consistent with previous studies involving various linear and structured oligonucleotide probes.

A linear DNA probe as an alternative to a molecular beacon for improving the sensitivity of a homogenous fluorescence biosensing platform for DNA detection using target-primed rolling circle amplification

Linear single-labeled DNA probes are used in this RCA-based fluorescence strategy for DNA detection, which could effectively avoid the fluorescence quenching between neighboring signal probes using hairpin probe as signal probe.

NanoCluster Beacons as reporter probes in rolling circle enhanced enzyme activity detection.

As a newly developed assay for the detection of endogenous enzyme activity at the single-catalytic-event level, Rolling Circle Enhanced Enzyme Activity Detection (REEAD) has been used to measure enzyme activity in both single human cells and malaria-causing parasites, Plasmodium sp. Current REEAD assays rely on organic dye-tagged linear DNA probes to report the rolling circle amplification products (RCPs), the cost of which may hinder the widespread use of REEAD. Here we show that a new class of activatable probes, NanoCluster Beacons (NCBs), can simplify the REEAD assays. Easily prepared without any need for purification and capable of large fluorescence enhancement upon hybridization, NCBs are cost-effective and sensitive. Compared to conventional fluorescent probes, NCBs are also more photostable. As demonstrated in reporting the human topoisomerases I (hTopI) cleavage-ligation reaction, the proposed NCBs suggest a read-out format attractive for future REEAD-based diagnostics.

Characterization of an electrochemical mercury sensor using alternating current, cyclic, square wave and differential pulse voltammetry

Here we report the characterization of an electrochemical mercury (Hg2+) sensor constructed with a methylene blue (MB)-modified and thymine-containing linear DNA probe. Similar to the linear probe electrochemical DNA sensor, the resultant sensor behaved as a “signal-off” sensor in alternating current voltammetry and cyclic voltammetry. However, depending on the applied frequency or pulse width, the sensor can behave as either a “signal-off” or “signal-on” sensor in square wave voltammetry (SWV) and differential pulse voltammetry (DPV). In SWV, the sensor showed “signal-on” behavior at low frequencies and “signal-off” behavior at high frequencies. In DPV, the sensor showed “signal-off” behavior at short pulse widths and “signal-on” behavior at long pulse widths. Independent of the sensor interrogation technique, the limit of detection was found to be 10nM, with a linear dynamic range between 10nM and 500nM. In addition, the sensor responded to Hg2+ rather rapidly; majority of the signal change occurred in <20min. Overall, the sensor retains all the characteristics of this class of sensors; it is reagentless, reusable, sensitive, specific and selective. This study also highlights the feasibility of using a MB-modified probe for real-time sensing of Hg2+, which has not been previously reported. More importantly, the observed “switching” behavior in SWV and DPV is potentially generalizable and should be applicable to most sensors in this class of dynamics-based electrochemical biosensors.

Thermodynamic Basis for Engineering High-Affinity, High-Specificity Binding-Induced DNA Clamp Nanoswitches

Naturally occurring chemoreceptors almost invariably employ structure-switching mechanisms, an observation that has inspired the use of biomolecular switches in a wide range of artificial technologies in the areas of diagnostics, imaging, and synthetic biology. In one mechanism for generating such behavior, clamp-based switching, binding occurs via the clamplike embrace of two recognition elements onto a single target molecule. In addition to coupling recognition with a large conformational change, this mechanism offers a second advantage: it improves both affinity and specificity simultaneously. To explore the physics of such switches we have dissected here the thermodynamics of a clamp-switch that recognizes a target DNA sequence through both Watson-Crick base pairing and triplex-forming Hoogsteen interactions. When compared to the equivalent linear DNA probe (which relies solely on Watson-Crick interactions), the extra Hoogsteen interactions in the DNA clamp-switch increase the probe's affinity for its target by ∼0.29 ± 0.02 kcal/mol/base. The Hoogsteen interactions of the clamp-switch likewise provide an additional specificity check that increases the discrimination efficiency toward a single-base mismatch by 1.2 ± 0.2 kcal/mol. This, in turn, leads to a 10-fold improvement in the width of the "specificity window" of this probe relative to that of the equivalent linear probe. Given these attributes, clamp-switches should be of utility not only for sensing applications but also, in the specific field of DNA nanotechnology, for applications calling for a better control over the building of nanostructures and nanomachines.

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.

Detection of Nucleic Acid Targets Using Ramified Rolling Circle DNA Amplification: A Single Nucleotide Polymorphism Assay Model

BackgroundIsothermal amplification methods provide alternatives to PCR that may be preferable for some nucleic acid target detection tasks. Among current isothermal target detection methods, ramified rolling circle amplification (RAM) of single-stranded DNA circles that are formed by ligation of linear DNA probes (C-probes or padlock probes) offers a unique target detection system by linked primers and a simple amplification system that is unconstrained by the target’s sequence context. Earlier implementations of RAM-based target detection were reported to be limited by background noise, due in part to unligated C-probe in the amplification reaction. We show here that a target-detection system using a biotinylated target-capture probe together with automated bead-handling reduces or eliminates background amplification noise. We demonstrate the system’s performance by detection of a single-nucleotide polymorphism in human genomic DNA.MethodologyTarget detection by RAM entails hybridization and ligation of a C-probe, followed by amplification and RAM signal detection. We evaluated RAM target detection in genomic DNA using recognition of a human Factor V gene single nucleotide polymorphism (G1691A) as a model. Locus-specific C-probes were annealed and ligated to genomic DNAs that represent the 3 possible genotypes at this locus, then ligated C-probes were amplified by real time RAM. The majority of the steps in the assay were performed with a magnetic bead-based chemistry on an automated platform. We show that the specificity of C-probe ligation permits accurate genotyping of this polymorphism. The assay as described here eliminates some of the background noise previously described for C-probe ligation, RAM amplification assays.ConclusionThe methods and results presented here show that a combination of C-probe detection, automated sample processing, and isothermal RAM amplification provide a practical approach for detecting DNA targets in complex mixtures.

Electroanalysis of single-nucleotide polymorphism by hairpin DNA architectures

Genetic analysis of infectious and genetic diseases and cancer diagnostics require the development of efficient tools for fast and reliable analysis of single-nucleotide polymorphism (SNP) in targeted DNA and RNA sequences often responsible for signalling disease onset. Here, we highlight the main trends in the development of electrochemical genosensors for sensitive and selective detection of SNP that are based on hairpin DNA architectures exhibiting better SNP recognition properties compared with linear DNA probes. SNP detection by electrochemical hairpin DNA beacons is discussed, and comparative analysis of the existing SNP sensing strategies based on enzymatic and nanoparticle signal amplification schemes is presented.

Single-labeled hairpin probe for highly specific and sensitive detection of lead(II) based on the fluorescence quenching of deoxyguanosine and G-quartet

Specific and homogeneous detection of heavy metal ion is of great importance for both human health care and environmental protection. We reported a highly specific and sensitive assay for fluorescent detection of Pb2+ based on the difference in quenching ability between deoxyguanosines and G-quartet by using carboxyfluorescein-labeled hairpin DNA (F-hpDNA) as a recognition probe. In the absence of target, the fluorescence of F-hpDNA can be quenched through photoinduced electron transfer from the dye to deoxyguanosines because the formation of hairpin brings deoxyguanosines close to the FAM. In the presence of Pb2+, the formation of G-quadruplex DNA leads to a significant decrease in fluorescence due to the effective stack of dye on the G-quartet, which obviously intensified the quenching of fluorophore. In comparison with linear DNA probe, hairpin DNA probe greatly improved the specificity, and Pb2+ can be highly selective detected even when coexisted with other metal ions. The quenching efficiency is linear with the concentration of lead(II) over the range of 0.5–500nM, with a limit of detection of 0.4nM. Conformational switch from hairpin to G-quadruplex was verified by CD measurements. Moreover, the application for detection of real samples further demonstrated its reliability. Therefore, it is a selective, simple and sensitive approach for detection of lead ion, as such, it promises to provide a solid foundation for developing universal analytical method for heavy metal ions.

Dendron-modified surfaces provide an ideal environment for stem–loop DNA probes

Specificity and sensitivity are important factors affecting DNA microarrays. Stem–loop DNA probes (SLPs) can be more specific in their recognition of target sequences than linear DNA probes, but unless they are carefully designed, surface interactions can disrupt the native stem–loop structure. In this study, we show how dendron-modified surfaces with well-defined, uniform spacing of aldehyde chemical functionalities offer an ideal substrate to immobilize SLPs and use them to detect nucleic acid targets. The mesospacing provided by the dendron-modified surfaces produces a solution-like environment that allows the SLPs to detect target nucleic acids at concentrations as low as 1pM in concentration.

An exonuclease III and graphene oxide-aided assay for DNA detection

We have developed a novel DNA assay based on exonuclease III (ExoIII)-induced target recycling and the fluorescence quenching ability of graphene oxide (GO). This assay consists of a linear DNA probe labeled with a fluorophore in the middle. Introduction of target sequence induces the exonuclease III catalyzed probe digestion and generation of single nucleotides. After each cycle of digestion, the target is recycled to realize the amplification. Finally, graphene oxide is added to quench the remaining probes and the signal from the resulting fluorophore labeled single nucleotides is detected. With this approach, a sub-picomolar detection limit can be achieved within 40min at 37°C. The method was successfully applied to multicolor DNA detection and the analysis of telomerase activity in extracts from cancer cells.