Beacon DNA Research Articles

Fluorescent aptasensors for sensitive detection of lead ions based on structure-switching DNA beacon probe and exonuclease I-mediated signal amplification

Herein, two simple fluorescent signal-on sensing strategies for detecting lead ions (Pb2+) were established based on structure-switching aptamer probes and exonuclease-assisted signal amplification strategies. Two hairpin-structure fluorescent probes with blunt-ended stem arms were designed by extending the base sequence of Pb2+ aptamer (PS2.M) and labelling the probes with FAM (in probe 1) and 2-aminopurine (2-AP) (in probe 2), respectively. In method 1, graphene oxide (GO) was added to adsorb probe 1 and quench the fluorescence emission of FAM to achieve low fluorescent background. In method 2, fluorescent 2-AP molecule inserted into the double-stranded DNA of probe 2 was quenched as a result of base stacking interactions, leading to a simplified, quencher-free approach. The addition of Pb2+ can induce the probes to transform into G-quadruplex structures, exposing single DNA strands at the 3′ end (the extended sequences). This exposure enables the activation of exonuclease I (Exo I) on the probes, leading to the cleavage effect and subsequent release of free bases and fluorophores, thereby resulting in amplified fluorescence signals. The two proposed methods exhibit good specificity and sensitivity, with detection limits of 0.327 nM and 0.049 nM Pb2+ for method 1 and method 2, respectively, and have been successfully applied to detect Pb2+ in river water and fish samples. Both detection methods employ the structure-switching aptamer probes and can be completed in two or three steps without the need for complex analytical instruments. Therefore, they have a broad prospect in the sensitive and simple detection of lead ion contamination in food and environmental samples.

Recent Advancements of Aptamers in Cancer Therapy.

Aptamers are chemical antibodies possessing the capability of overcoming the limitations posed by conventional antibodies, particularly for diagnostic, therapeutic, and theranostic applications in cancer. The ease of chemical modifications or functionalization, including conjugations with nucleic acids, drug molecules, and nanoparticles, has made these aptamers to gain priorities in research. In this Mini-review, various reports on therapeutics with aptamer-functionalized nanomaterials for controlled or multistep drug release, targeted delivery, stimuli-responsive drug release, etc. are discussed. In the case of nucleic-acid-conjugated aptamers, DNA nanotrains and DNA beacons are discussed in terms of the possibility of multidrug loading for chemotherapy and gene therapy. Developments with electrochemical aptasensors and signal-enhanced immune aptasensors are also discussed. Further, the future scope of aptamer technology in cancer theranostics and the prevailing limitations are discussed.

Detection of E.coli 23S rRNA by electrocatalytic “off-on” DNA beacon assay with femtomolar sensitivity

Prevention of food spoilage, environmental bio-contamination, and pathogenic infections requires rapid and sensitive bacterial detection systems. Among microbial communities, the bacterial strain of Escherichia coli is most widespread, with pathogenic and non-pathogenic strains being biomarkers of bacterial contamination. Here, we have developed a fM-sensitive, simple, and robust electrocatalytically-amplified assay facilitating specific detection of E.coli 23S ribosomal rRNA, in the total RNA sample, after its site-specific cleavage by RNase H enzyme. Gold screen-printed electrodes (SPE) were electrochemically pre-treated to be productively modified with a methylene-blue (MB) – labelled hairpin DNA probes, which hybridization with the E. coli-specific DNA placed MB in the top region of the DNA duplex. The formed duplex acted as an electrical wire, mediating electron transfer from the gold electrode to the DNA-intercalated MB, and further to ferricyanide in solution, enabling its electrocatalytic reduction otherwise impeded on the hairpin-modified SPEs. The assay facilitated 20 min 1 fM detection of both synthetic E. coli DNA and 23S rRNA isolated from E.coli (equivalent to 15 CFU mL−1), and can be extended to fM analysis of nucleic acids isolated from any other bacteria.

Highly Accurate and Fast Electrochemical Detection of Scrub Typhus DNA via a Nanoflower NiFe-Based Biosensor.

Owing to the lack of specific diagnostic methods, Scrub typhus can sometimes be difficult to diagnose in the Asia-Pacific region. Therefore, an efficient and rapid detection method urgently needs to be developed. Based on competitive single-stranded DNA over modified glassy carbon electrode (GCE), an electrochemical biosensor was established to detect the disease. The nano-flower NiFe layered double hydroxide (NiFe-LDH) modified GCE has a large specific surface area, which supported a large amount of gold nanoparticles, so that a wide linear detection range from 25 fM to 0.5 μM was obtained. The beacon DNA (B-DNA) with the same sequence as the Scrub typhus DNA (T-DNA), but labeled with methylene blue, was used to construct a competitive relationship. When T-DNA and B-DNA were present on the sensor simultaneously, they would hybridize with probe DNA in a strong competition, and the corresponding electrochemical response signal would be generated via testing. It contributed to reducing tedious experimental procedures and excessive response time, and achieved fast electrochemical detection of DNA. The strategy provides a worthy avenue and possesses promising applications in disease diagnosis.

Short DNA Oligonucleotide as a Ag+ Binding Detector.

Ag+ has been known to mediate several natural metallo-base pairs. Based on the unique structural information of a short 8-mer DNA strand (5′-GCACGCGC-3′) induced by Ag+, we constructed several fluorescent DNA beacons for the detection of Ag+ according to the increase in the fluorescence emission on Ag+ binding. This Ag+ detection assay is quick, sensitive, and easy to adapt and can function in a wide range of temperatures from 5 to 65 °C.

Graphene setting the stage: tracking DNA hybridization with nanoscale resolution

In this study we use nanophotonic effects of graphene to study DNA hybridization: the z−4 nanoscale distance-dependence of the fluorescence lifetime for fluorophores located in the vicinity of graphene is for the first time used to track a DNA hybridization reaction with nanoscale resolution in real time. First, a nanostaircase with ≈2 nm steps from 0 to a total height of 48 nm is used as a nanoruler to confirm the distance dependence law. We find that the axial sensitivity is suited to determine the nanoscale surface roughness of these samples. The proof-of-concept DNA experiments in aqueous medium involve the hybridization of fluorescently labelled DNA beacons attached to CVD grown graphene with complementary (target) DNA added in solution. We track the conformational changes of the beacons statistically by determining the fluorescence lifetimes of the labelling dye and converting them into nanoscale distances from the graphene. In this way, we are able to monitor the vertical displacement of the label during DNA-beacon unfolding with an axial resolution reaching down to 1 nm. The measured distance increase during the DNA hybridization reaction of about 10 nm matches the length of the target DNA strand. Furthermore, the width of the fluorescence lifetime distributions could be used to estimate the molecular tilt angle of the hybridized ds-DNA configuration. The achieved nanoscale sensitivity opens innovation opportunities in material engineering, genetics, biochemistry and medicine.

A NIR Light Gated DNA Nanodevice for Spatiotemporally Controlled Imaging of MicroRNA in Cells and Animals.

Nanodevices have potential as intelligent sensing systems for detection of microRNAs (miRNAs) in living cells. However, the resolution offered by "always active" nanodevices is often insufficient to manipulatemiRNAsensing with high spatiotemporal control. In this work, using DNA nanotechnology we constructed an activatable DNA nanodevice programmed to detect miRNAs in vitro and in vivo with the high spatial and temporal precision of NIR light. Our nanodevice is functionalized on the surface of upconversion nanoparticles (UCNPs) with a rationally designed DNA beacon that displays UV light-activatable miRNA sensing activity. The UCNPs absorb deep-tissue-penetrable NIR light and emit high-energy UV light locally, which serve as transducers to operate the nanodevice in the NIR window. The nanodevice can naturally enter cells and enable remote regulation of its fluorescent imaging activity for miRNAs in living cells by NIR light illumination in a chosen place and time. Furthermore, we demonstrate that the nanodevice can be expanded to activatable imaging of intratumoral miRNAs in living mice. This work illustrates the potential of DNA nanodevices for miRNA detection with high spatiotemporal resolution, which could expand the toolbox of technologies for precise biological and medical analysis.

Fluorometric determination of nucleic acids based on the use of polydopamine nanotubes and target-induced strand displacement amplification.

The authors describe a fluorometric method for the quantitation of nucleic acids by combining (a) cycled strand displacement amplification, (b) the unique features of the DNA probe SYBR Green, and (c) polydopamine nanotubes. SYBR Green undergoes strong fluorescence enhancement upon intercalation into double-stranded DNA (dsDNA). The polydopamine nanotubes selectively adsorb single-stranded DNA (ssDNA) and molecular beacons. In the absence of target DNA, the molecular beacon, primer and SYBR Green are adsorbed on the surface of polydopamine nanotubes. This results in quenching of the fluorescence of SYBR Green, typically measured at excitation/emission wavelengths of 488/518nm. Upon addition of analyte (target DNA) and polymerase, the stem of the molecular beacon is opened so that it can bind to the primer. This triggers target strand displacement polymerization, during which dsDNA is synthesized. The hybridized target is then displaced due to the strand displacement activity of the polymerase. The displaced target hybridizes with another molecular beacon. This triggers the next round of polymerization. Consequently, a large amount of dsDNA is formed which is detected by addition of SYBR Green. Thus, sensitive and selective fluorometric detection is realized. The fluorescent sensing strategy shows very good analytical performances towards DNA detection, such as a wide linear range from 0.05 to 25nM with a low limit of detection of 20 pM. Graphical abstract Schematic of a fluorometric strategy for highly sensitive and selective determination of nucleic acids by combining strand displacement amplification and the unique features of SYBR Green I (SG) and polydopamine nanotubes.

Wafer scale fabrication of graphene microelectrode arrays for the detection of DNA hybridization

Since the discovery of graphene, and due to its unique properties, we have witnessed a growing interest in the use of graphene-based devices for applications in the most diverse areas, namely in biosensing, particularly in the detection of genetic material. DNA can bind directly to graphene without the need of a linker and that makes this platform highly interesting for biosensor development.Here, electrochemical chips consisting of 6 independent gold microelectrode arrays as working electrode, and platinum reference and counter electrodes were fabricated at the wafer scale and, after graphene transfer and patterning, were used in the detection of DNA hybridization. Combining the sensitivity of electrochemical impedance spectroscopy and the selectivity of DNA beacons, we were able to detect DNA hybridization in a linear range between 5pM and 5nM, which is in the relevant clinical range for many diseases, with sensitivity to single nucleotide polymorphism.

Effect of a Dual Charge on the DNA-Conjugated Redox Probe on DNA Sensing by Short Hairpin Beacons Tethered to Gold Electrodes.

Charges of redox species can critically affect both the interfacial state of DNA and electrochemistry of DNA-conjugated redox labels and, as a result, the electroanalytical performance of those systems. Here, we show that the kinetics of electron transfer (ET) between the gold electrode and methylene blue (MB) label conjugated to a double-stranded (ds) DNA tethered to gold strongly depend on the charge of the MB molecule, and that affects the performance of genosensors exploiting MB-labeled hairpin DNA beacons. Positively charged MB binds to dsDNA via electrostatic and intercalative/groove binding, and this binding allows the DNA-mediated electrochemistry of MB intercalated into the duplex and, as a result, a complex mode of the electrochemical signal change upon hairpin hybridization to the target DNA, dominated by the "on-off" signal change mode at nanomolar levels of the analyzed DNA. When MB bears an additional carboxylic group, the negative charge provided by this group prevents intimate interactions between MB and DNA, and then the ET in duplexes is limited by the diffusion of the MB-conjugated dsDNA (the phenomenon first shown in Farjami , E. ; Clima , L. ; Gothelf , K. ; Ferapontova , E. E. Anal. Chem. 2011 , 83 , 1594 ) providing the robust "off-on" nanomolar DNA sensing. Those results can be extended to other intercalating redox probes and are of strategic importance for design and development of electrochemical hybridization sensors exploiting DNA nanoswitchable architectures.

High DNA melting activity of extremophyte Eutrema salsugineum cold shock domain proteins EsCSDP1 and EsCSDP3

Plant cold shock domain proteins (CSDP) participate in maintenance of plant stress tolerance and in regulating their development. In the present paper we show that two out of three extremophyte plant Eutrema salsugineum proteins EsCSDP1-3, namely EsCSDP1 and EsCSDP3, possess high DNA-melting activity. DNA-melting activity of proteins was evaluated using molecular beacon assay in two ways: by measuring Tm parameter (the temperature at which half of the DNA beacon molecules is fully melted) and the beacon fluorescence at 4°C. As the ratio protein/beacon was increased, a decrease in Tm was observed. Besides DNA-melting activity of full proteins, activity was measured for three isolated cold shock domains EsCSD1-3, C-terminal domain of EsCSDP1 (EsZnF1), as well as a mixture of EsCSD1 and EsZnF1. The Tm reduction efficiency of proteins formed the following sequence: EsCSDP3≈EsCSDP1>(EsCSD1+EsZnF1)>EsZnF1>EsCSDP2. Only full proteins EsCSDP3 and EsCSDP1 demonstrated DNA-melting activity at 4°C. The presented experimental data indicate that i: interaction of EsCSDP1-3 with beacon single-stranded region is obligatory for efficient melting; ii: cold shock domain and C-terminal domain with zinc finger motifs should be present in one protein molecule to have high melting activity.

High-throughput bead-based identification of structure-switching aptamer beacons.

We describe a new platform to identify structure-switching DNA beacon aptamers, which detect small molecules in a specific manner. By clonally amplifying a DNA library designed to fluoresce in response to binding events onto microbeads, aptamer beacons can be selected by stringent fluorescence-assisted sorting. We validated this method by isolating known and novel anti-steroid aptamers from two separate DNA libraries that were structurally enriched with three-way junctions. Importantly, aptamers were retrieved in only a few (three) rounds of selection by this approach and did not require further optimization, significantly streamlining the process of beacon development.

PNA-Induced Assembly of Fluorescent Proteins Using DNA as a Framework

Controlled alignment of proteins on molecular frameworks requires the development of facile and orthogonal chemical approaches and molecular scaffolds. In this work, protein-PNA conjugates are brought forward as new chemical components allowing efficient assembly and alignment on DNA scaffolds. Site-selective monomeric teal fluorescent protein (mTFP)-peptide nucleic acid (PNA) (mTFP-PNA) conjugation was achieved by covalent linkage of the PNA to the protein through expressed protein ligation (EPL). A DNA beacon, with 6-Fam and Dabcyl at its ends, acts as a framework to create an assembled hetero-FRET system with the mTFP-PNA conjugate. Using fluorescence intensity, frequency domain lifetime measurements, and anisotropy measurements, the system was shown to produce FRET as indicated by decreased donor intensity, decreased donor lifetime, and increased donor anisotropy. Extension of the DNA scaffold allowed for the assembly of multiple mTFP-PNA constructs. Efficient formation of protein dimers and oligomers on the DNA-PNA frameworks could be shown, as visualized via size exclusion chromatography (SEC) and electrophoresis (SDS-PAGE). Assembly of multiple proteins in a row induced homo-FRET for the mTFP-PNA's assembled on the DNA scaffolds. The oligonucleotide framework allows an induced and controllable assembly of proteins by fusing them to PNAs directed to align on DNA scaffolds.

A novel probe density controllable electrochemiluminescence biosensor for ultra-sensitive detection of Hg2+ based on DNA hybridization optimization with gold nanoparticles array patterned self-assembly platform

Biosensor based on DNA hybridization holds great potential to get higher sensitivity as the optimal DNA hybridization efficiency can be achieved by controlling the distribution and orientation of probe strands on the transducer surface. In this work, an innovative strategy is reported to tap the sensitivity potential of current electrochemiluminescence (ECL) biosensing system by dispersedly anchoring the DNA beacons on the gold nanoparticles (GNPs) array which was electrodeposited on the glassy carbon electrode surface, rather than simply sprawling the coil-like strands onto planar gold surface. The strategy was developed by designing a “signal-on” ECL biosensing switch fabricated on the GNPs nanopatterned electrode surface for enhanced ultra-sensitivity detection of Hg2+. A 57-mer hairpin-DNA labeled with ferrocene as ECL quencher and a 13-mer DNA labeled with Ru(bpy)32+ as reporter were hybridized to construct the signal generator in off-state. A 31-mer thymine (T)-rich capture-DNA was introduced to form T–T mismatches with the loop sequence of the hairpin-DNA in the presence of Hg2+ and induce the stem-loop open, meanwhile the ECL “signal-on” was triggered. The peak sensitivity with the lowest detection limit of 0.1nM was achieved with the optimal GNPs number density while exorbitant GNPs deposition resulted in sensitivity deterioration for the biosensor. We expect the present strategy could lead the renovation of the existing probe-immobilized ECL genosensor design to get an even higher sensitivity in ultralow level of target detection such as the identification of genetic diseases and disorders in basic research and clinical application.

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.

“Off−On” Electrochemical Hairpin-DNA-Based Genosensor for Cancer Diagnostics

A simple and robust "off-on" signaling genosensor platform with improved selectivity for single-nucleotide polymorphism (SNP) detection based on the electronic DNA hairpin molecular beacons has been developed. The DNA beacons were immobilized onto gold electrodes in their folded states through the alkanethiol linker at the 3'-end, while the 5'-end was labeled with a methylene blue (MB) redox probe. A typical "on-off" change of the electrochemical signal was observed upon hybridization of the 27-33 nucleotide (nt) long hairpin DNA to the target DNA, in agreement with all the hitherto published data. Truncation of the DNA hairpin beacons down to 20 nts provided improved genosensor selectivity for SNP and allowed switching of the electrochemical genosensor response from the on-off to the off-on mode. Switching was consistent with the variation in the mechanism of the electron transfer reaction between the electrode and the MB redox label, for the folded beacon being characteristic of the electrochemistry of adsorbed species, while for the "open" duplex structure being formally controlled by the diffusion of the redox label within the adsorbate layer. The relative current intensities of both processes were governed by the length of the formed DNA duplex, potential scan rate, and apparent diffusion coefficient of the redox species. The off-on genosensor design used for detection of a cancer biomarker TP53 gene sequence favored discrimination between the healthy and SNP-containing DNA sequences, which was particularly pronounced at short hybridization times.

The RNA Chaperone Hfq Makes a Transient Ternary Complex with RNA Strands to Facilitate RNA Annealing

In bacteria, small anti-sense RNAs (sRNAs) help control the cell's response to environmental stress by binding complementary sequences in their mRNA targets. Hfq protein is required for the regulatory function of anti-sense sRNAs, and facilitates the formation of sRNA-mRNA complexes. Hfq is a ring-shaped homohexamer that preferentially binds single-stranded U-rich RNA on its proximal face and A-rich RNA on its distal face. Although Hfq has been shown to accelerate RNA base pairing and strand exchange in vitro, how Hfq does this is still unknown. To model the annealing mechanism of Hfq, we used a U-rich16-nt RNA (D16). Stopped-flow FRET measurements using fluorescein-labeled D16 RNA and Cy3-labeled Hfq showed that Hfq binds single-stranded RNA rapidly (≥ 7•107M−1s−1). Upon the addition of a complementary RNA, Hfq is rapidly released from the RNA complex (kobs ∼ 30 s−1). Stopped-flow experiments with a fluorescent molecular beacon showed that Hfq accelerates RNA annealing and strand exchange one hundred times. However, annealing is slower than Hfq release, suggesting the protein dissociates before the duplex is fully formed. Hfq mutations and competition experiments suggest that D16 RNA and the RNA beacon interact with the proximal and distal faces of Hfq, respectively, as expected. When a DNA beacon that binds Hfq weakly (1.4 μM) is used instead of an RNA beacon, Hfq-mediated RNA annealing is faster, but saturates at a higher concentration of Hfq. These results are consistent with rapid binding of RNA to the distal and proximal faces of Hfq, forming a transient ternary complex. We propose that Hfq leaves the complex before RNA hybridization is complete, after which the RNA strands form a stable duplex or dissociate and rebind Hfq as single-strands for the next round of annealing.

Detection of an abasic site in RNA with stem-loop DNA beacons: Application to an activity assay for Ricin Toxin A-Chain

The catalytic ability of Ricin Toxin A-Chain (RTA) to create an abasic site in a 14-mer stem-tetraloop RNA is exploited for its detection. RTA catalyzes the hydrolysis of the N-glycosidic bond of a specific adenosine in the G A GA tetraloop of stem-loop RNA. Thus, a 14-mer stem-loop RNA substrate containing an intact “G A GA” sequence can be discriminated from the product containing an abasic “G ab GA” sequence by hybridization with a 14-mer DNA stem-loop probe sequence and following the fluorescent response of the heteroduplexes. Three DNA beacon probe designs are described. Beacon 1 probe is a stem-loop structure and has a fluorophore and a quencher covalently linked to the 5′- and 3′-ends. In this format the probe–substrate heteroduplex gives a fluorescent signal while the probe–product one remains quenched. Beacon 2 is a modified version of 1 and incorporates a pyrene deoxynucleoside for recognition of the abasic site. In this format both the substrate and product heteroduplexes give a fluorescent response. Beacon 3 utilizes a design where the fluorophore is on the substrate RNA sequence at its 5′-end while the quencher is on the probe DNA sequence at its 3′-end. In this format the fluorescence of the substrate–probe heteroduplex is quenched while that of the product–probe one is enhanced. The lower limit of detection with beacons is 14 ng/mL of RTA.

Modeling DNA beacons at the mesoscopic scale

We report model calculations on DNA single strands which describe the equilibrium dynamics and kinetics of hairpin formation and melting. Modeling is at the level of single bases. Strand rigidity is described in terms of simple polymer models; alternative calculations performed using the freely rotating chain and the discrete Kratky-Porod models are reported. Stem formation is modeled according to the Peyrard-Bishop-Dauxois Hamiltonian. The kinetics of opening and closing is described in terms of a diffusion-controlled motion in an effective free-energy landscape. Melting profiles, dependence of melting temperature on loop length, and kinetic time scales are in semiquantitative agreement with experimental data obtained from fluorescent DNA beacons forming poly(T) loops. Variation in strand rigidity is not sufficient to account for the large activation enthalpy of closing and the strong loop length dependence observed in hairpins forming poly(A) loops. Implications for modeling single strands of DNA or RNA are discussed.

Single-Strand Stacking Free Energy from DNA Beacon Kinetics

DNA beacons are short single-stranded chains which can form closed hairpin shapes through complementary base pairing at their ends. Contrary to the common polymer theory assumption that only their loop length matters, experiments show that their closing kinetics depend on the loop composition. We have modeled the closing kinetics and in so doing have obtained stacking enthalpies and entropies for single-stranded nucleic acids. The resulting change of persistence length with temperature effects the dynamics. With a Monte Carlo study, we answer another polymer question of how the closing time scales with chain length, finding tau approximately N(2.44+/-0.02). There is a significant crossover for shorter chains, bringing the effective exponent into good agreement with experiment.