Molecular Beacon DNA Research Articles

AFM-Imaging Diagnosis Method for Single Nucleotide Polymorphism Using Molecular Beacon DNA as an Intramolecular Ligation Template of Target DNA and a Viewable Indicator

An AFM-imaging-based method for single nucleotide polymorphism (SNP) analysis is described. A stem-loop-forming 34-mer oligonucleotide (p34s) was designed. p34s contains the complementary sequence for K-ras (5'-GGT GGC-3', t6G), one of the human oncogenes, at the 5'-end for target-recognition and five successive phosphorothioate linkages in the loop. The functional probe, either alone or hybridized with target DNA (p34s/t6G), relaxed upon treatment with "opener" DNA. The template/target DNA interstrand hybridization product is covalently connected by ligase if the correct target is used, but not hybridized species including mismatches. With these results, developed was a solid-phase SNP assay by transferring an aliquot of the product onto an Au(111) substrate for self-assembly, followed by AFM imaging. Clear contrasts that allow the detection of SNPs, were observed for the ligated and non-ligated species representing the loop-to-linear conformational change. Simple statistical surface-roughness analysis determined the lowest concentration of the sample to be 5 × 10(-10) M, whose necessary sample quantity was 5 fmol.

Exonuclease-assisted cascaded recycling amplification for label-free detection ofDNA

The network consisting of three kinds of unlabeled stem-loop DNA molecular beacons (MBs) is activated by target DNA in the presence of exonuclease-III (Exo-III), achieving the concept of exonuclease-assisted cascaded recycling amplification (Exo-CRA) for DNA detection with a wide dynamic range of 8 orders of magnitude.

Synthesis and Properties of Molecular Beacon DNA Probe Bearing Novel Silylated Pyrene Derivative

A novel stem-loop structured fluorescent oligoDNA probe (molecular beacon probe) bearing a silylated pyrene derivative at C-5 position of deoxyuridine has been synthesized. The fluorescently labeled modified nucleoside has been incorporated into two consecutive positions in the stem segment of the DNA using an automated DNA synthesizer. The resulting modified DNA exhibited an excimer fluorescent signal upon binding to the fully matched complementary DNA strand. The excimer emission was, however, effectively quenched while it stays alone or it hybridizes to a single base mismatched complementary target.

Electrochemical molecular beacon DNA biosensor for the detection and discrimination of the DF508 cystic fibrosis mutation

Cystic fibrosis is one of the most common genetically inherited diseases in Northern Europe, consisting of a defect of chloride transport in the epithelium, with the DF508 mutation being the most common mutation associated with the disease. In this work the design and characterisation of a reagent-less electrochemical genosensor, based on the use of an electrochemical molecular beacon targeting the DF508 mutation is presented. Different aspects of the sensing platform including molecular beacon design and surface chemistry of the sensor surface were evaluated. Operational parameters such as assay buffer and assay time were also optimised. Using the optimised molecular beacon designs a clear differentiation between the targeted sequence (i.e. mutant) and potential interferent (i.e. wild type) was demonstrated, with a total required assay time of 20 min. The major advantage of the proposed reagent-less sensing platform is the fact that this only required, as intervention of the end-user, the addition of the sample.

Abstract 3062: A comparative analysis of phosphothioated DNA and peptide nucleic acid molecular beacons targeting specific K-ras mutation

Abstract Introduction: Early detection of gastrointestinal malignancies may improve disease-related outcome. In vivo, real time imaging using hybridization of fluorescently-labeled complementary transcripts to intracellular RNA sequences may facilitate early detection of malignant and pre-malignant lesions Methods: Using K-ras oncogene as a model target, and adapting the molecular beacon technology, we performed an hybridization kinetics and hybridization specificity comparison of two types of molecular beacons (MBs) based on either phosphothioated DNA (PS-DNA-MB) or peptide nucleic acid (PNA-TO-MB, where thiazole orange [TO] was used as the fluorescent molecule) both in vitro and in a variety of cell lines. In order to determine the sequence-specificity of MB's at a single base resolution, a codon 12 K-ras oncogene mutation (GGT→GAT) was used as target in different cell lines. Both PS-DNA and PNA-TO MB's were designed to target this specific K-ras point mutation. We studied the hybridization of the two MBs to wild-type K-ras (HT-29 colon cancer cell line) and two cell lines, one harboring this K-ras point mutation (PANC-1) and SW-480 harboring a different (GGT→GTT) mutation. Results: Hybridization kinetics of PNA-TO-MB to target DNA or RNA was faster compared to PS-DNA-MB highlighting the advantage of such probes to be developed into diagnostic kits for the early detection of cancer. However, in solution, using synthetic DNA oligomers as targets, PS-DNA-MB demonstrated higher specificity to its complementary target (3-to 4.6-fold increase in fluorescence in comparison to DNA with a single mismatch) compared to the PNA-TO-MB (only 1.3- to 2.2-fold increase). Yet, incubation of PNA-TO-MB with total RNA (isolated from these cell lines) and with fixed cells, resulted in a significantly higher fluorescent signal (p < 0.001) detected for target (fully complement, PANC-1) compared to single mismatch mRNA transcript in HT29 and SW-480. In contrast, the PS-DNA-MB showed poor discrimination in total RNA and no fluorescent signal with all cell lines after 5 minutes incubation. Conclusions: Based on the fast hybridization kinetics and based on the single mismatch discrimination found for PNA-TO-MB in fixed cells and with total RNA, it is speculated that such PNA-TO-MB's are promising candidates for the early detection of cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3062. doi:10.1158/1538-7445.AM2011-3062

Locked nucleic acid hairpin detection of UV-induced DNA damage

The exposure of DNA to radiation and chemical insults leads to damage and disease. Thus, detection and understanding of DNA damage is important for elucidating molecular mechanisms of disease. However, current methods of DNA damage detection are either time-consuming, destroy the sample, or are too specific to be used for generic detection of damage. In this paper, we examine the sensitivity of different locked nucleic acid (LNA) hairpin probes for detecting UV-induced oligonucleotide damage to LNA composition, target concentration, and ionic strength. We also compare the selectivity of the LNA hairpin probe with a DNA molecular beacon (MB) hairpin probe for detecting DNA damage. Our results show that the selectivity of the LNA hairpin probe to UV-induced nucleic acid damage increases with increasing ionic strength of the buffer and decreases with increasing target concentration and with increasing LNA ratio.

“Molecular Beacon”-Based Fluorescent Assay for Selective Detection of Glutathione and Cysteine

We report on the development of a fluorescence turn-on "molecular beacon" probe for the detection of glutathione (GSH) and cysteine (Cys). The method is based on a competitive ligation of Hg(2+) ions by GSH/Cys and thymine-thymine (T-T) mismatches in a DNA strand of the self-hybridizing beacon strand. The assay relies on the distance-dependent optical properties of the fluorophore/quencher pair attached to the ends of the molecular beacon DNA strand. In a very selective coordination of Hg(2+) to GSH/Cys, the fluorophore/quencher distance increases concomitantly with the dehybridization and dissociation of the beacon stem T-Hg(2+)-T due to the extraction of Hg(2+) ions. This process results in switching the molecular beacon to the "on" state. The concentration range of the probe is 4-200 nM with the limit of detection (LOD) of 4.1 nM for GSH and 4.2 nM Cys. The probe tested satisfactorily against interference for a range of amino acids including sulfur-containing methionine.

Hairpin DNA-functionalized gold colloids for the imaging of mRNA in live cells.

A strategy is presented for the live cell imaging of messenger RNA using hairpin DNA-functionalized gold nanoparticles (hAuNP). hAuNP improve upon technologies for studying RNA trafficking by their efficient internalization within live cells without transfection reagents, improved resistance to DNase degradation, low cytotoxicity, and the incorporation of hairpin DNA molecular beacons to confer high specificity and sensitivity to the target mRNA sequence. Furthermore, the targeted nanoparticle-beacon construct, once bound to the target mRNA sequence, remains hybridized to the target, enabling spatial and temporal studies of RNA trafficking and downstream analysis. Targeted hAuNP exhibited high specificity for glyceraldehyde 3-phosphate dehydrogenase (GADPH) mRNA in live normal HEp-2 cells and respiratory syncytial virus (RSV) mRNA in live RSV-infected HEp-2 cells with high target to background ratios. Multiplexed fluorescence imaging of distinct mRNAs in live cells and simultaneous imaging of mRNAs with immunofluorescently stained protein targets in fixed cells was enabled by appropriate selection of molecular beacon fluorophores. Pharmacologic analysis suggested that hAuNP were internalized within cells via membrane-nanoparticle interactions. hAuNP are a promising approach for the real-time analysis of mRNA transport and processing in live cells for elucidation of biological processes and disease pathogenesis.

DNA interactions with a Methylene Blue redox indicator depend on the DNA length and are sequence specific

A DNA molecular beacon approach was used for the analysis of interactions between DNA and Methylene Blue (MB) as a redox indicator of a hybridization event. DNA hairpin structures of different length and guanine (G) content were immobilized onto gold electrodes in their folded states through the alkanethiol linker at the 5'-end. Binding of MB to the folded hairpin DNA was electrochemically studied and compared with binding to the duplex structure formed by hybridization of the hairpin DNA to a complementary DNA strand. Variation of the electrochemical signal from the DNA-MB complex was shown to depend primarily on the DNA length and sequence used: the G-C base pairs were the preferential sites of MB binding in the duplex. For short 20 nts long DNA sequences, the increased electrochemical response from MB bound to the duplex structure was consistent with the increased amount of bound and electrochemically readable MB molecules (i.e. MB molecules that are available for the electron transfer (ET) reaction with the electrode). With longer DNA sequences, the balance between the amounts of the electrochemically readable MB molecules bound to the hairpin DNA and to the hybrid was opposite: a part of the MB molecules bound to the long-sequence DNA duplex seem to be electrochemically mute due to long ET distance. The increasing electrochemical response from MB bound to the short-length DNA hybrid contrasts with the decreasing signal from MB bound to the long-length DNA hybrid and allows an "off"-"on" genosensor development.

A novel single nucleotide polymorphism detection of a double-stranded DNA target by a ribonucleotide-carrying molecular beacon and thermostable RNase HII

Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation. SNPs are important markers that link sequence variations to phenotypic changes. Because of the importance of SNPs in the life and medical sciences, a great deal of effort has been devoted to developing accurate, rapid, and cost-effective technologies for SNP analysis. In this article, we describe a novel method for SNP genotyping based on differential fluorescence emission due to cleavage by Thermus thermophilus RNase HII (TthRNase HII) of DNA heteroduplexes containing an SNP site-specific chimeric DNA–rN 1–DNA molecular beacon (cMB). We constructed a loop sequence for a cMB that contains a single SNP-specific ribonucleotide at the central site. When the cMB probe is hybridized to a target double-stranded DNA (dsDNA), a perfect match of the cMB/DNA duplex permits efficient cleavage with TthRNase HII, whereas a mismatch in the duplex due to an SNP greatly reduces efficiency. Cleavage efficiency is measured by the incremental difference of fluorescence emission of the beacon. We show that the genotypes of 10 individuals at 12 SNP sites across a series of human leukocyte antigen (HLA) can be determined correctly with respect to conventional DNA sequencing. This novel TthRNase HII-based method offers a platform for easy and accurate SNP analysis.

A molecular beacon approach to detecting RAD52 expression in response to DNA damage in human cells

Although DNA damage proteins are infrequently regulated at the transcriptional level, RAD52 mRNA levels appear to be significantly induced in human cells following methyl methanesulphonate (MMS) and Etoposide treatment. Studies have so far been limited to biochemical analysis of cellular extracts and we aimed to extend this observation to whole cells. To address this, we have developed a series of molecular beacon (MB) probes that fluoresce upon hybridising with RAD52 mRNA sequence. MB’s are synthetic hairpin probes, which generate a significant fluorescent signal only upon hybridising complementary nucleotide. Three MB’s are described herein, which display differential sensitivity, specificity and stability. In particular, the suitability of a texas red-labelled DNA MB (TR-MB), a dual-labelled (FAM-TAMRA) fluorescence resonance energy transfer-capable DNA MB (FRET-MB) and a FAM-labelled MB of 2′-O-methylated RNA backbone (FAM-MB) was investigated. We conclude that FAM-MB is most suitable for intracellular applications, and demonstrate a positive correlation between MB fluorescence intensity, RAD52 gene expression and both gamma ionising radiation and MMS concentration in human TK6 cells. RAD52 contribution to DNA repair has been ascribed to its role in homologous recombination (HR) and therefore we propose FAM-MB could be a potential tool for discriminating between substrates of HR and non-homologous end joining (NHEJ).

Design of Molecular Beacons as Signaling Probes for Adenosine Triphosphate Detection in Cancer Cells Based on Chemiluminescence Resonance Energy Transfer

In the present study, binary and triplex DNA molecular beacons, as signaling probes based on a luminol-H(2)O(2)-horseradish peroxidase (HRP)-fluorescein chemiluminescence resonance energy transfer (CRET) system and structure-switching aptamers for highly sensitive detection of small molecules, are developed using adenosine triphosphate (ATP) as a model analyte to demonstrate the generality of the strategy. This CRET process occurs from donor luminol to acceptor fluorescein, which is oxidized by H(2)O(2) and catalyzed by HRP. DNA aptamer for ATP is first attached on the surface of magnetic nanoparticles (MNPs). The cDNA linker has an extension that hybridizes with two other DNAs (LumAuNP-DNA and F-DNA) or three other DNAs (HRP-DNA, LumAuNP-DNA, and F-DNA) to fabricate CRET-BMBP-MNP or CRET-TMBP-MNP conjugates that provide the CRET signals. Thus, in the absence of ATP, when the MNPs are removed from the solution, they also take with them the linker DNA and the CRET signal probes, and no CRET signal can be detected. However, when ATP is introduced, a competition for the ATP aptamer between ATP and the cDNA linker occurs. As a result, CRET-BMBP and CRET-TMBP are forced to dissociate from the MNP surface based on the structure switching of the aptamer. The CRET signals are proportional to the concentration of ATP. In order to accelerate the rate of the aptamer structure-switching process, an invader DNA is introduced into the proposed strategy. The present CRET system provides a low detection limit of 1.1 x 10(-7) and 3.2 x 10(-7) M for ATP detection by BMBP and TMBP, respectively, which also exhibits a good selectivity for ATP detection. Sample assays of ATP in K562 leukemia cells and 4T1 breast cancer cells confirm the reliability and practicality of the protocol, which reveal a good prospect of this platform for biological sample analysis.

The use of DNA molecular beacons as nanoscale temperature probes for microchip-based biosensors

Today's biosensors and drug delivery devices are increasingly incorporating lithographically patterned circuitry that is placed within microns of the biological molecules to be detected or released. Elevated temperatures due to Joule heating from the underlying circuitry cannot only reduce device performance, but also alter the biological activity of such molecules (i.e. binding, enzymatic, folding). As a consequence, biochip design and characterization will increasingly require local measurements of the temperature and temperature gradients on the biofunctionalized surface. We have developed a technique to address this challenge based on the use of DNA molecular beacons as a nanoscale temperature probe. The surface of fused-silica chips with lithographically patterned, current-carrying gold rings have been functionalized with a layer of molecular beacons. We utilize the temperature dependence of the molecular beacons to calibrate the temperature at the center of the rings as a function of applied current from 25 to 50 °C. The fluorescent images of the rings reveal the extent of heating to the surrounding chip due to the applied current while resolving temperature gradients over length scales of less than 500 nm. Finite element analysis and analytic calculations of the distribution of heat in the vicinity of the current-carrying rings agree well with the experimental results. Thus, molecular beacons are shown to be a viable tool for temperature calibration of micron-sized circuitry and the visualization of submicron temperature gradients.

A Controllable Solid-State Ru(bpy)32+ Electrochemiluminescence Film Based on Conformation Change of Ferrocene-Labeled DNA Molecular Beacon

A controllable solid-state electrochemiluminescence (ECL) film based on efficient and stable quenching of ECL of ruthenium(II) tris-(bipyridine) (Ru(bpy)32+) by oxidizing ferrocene (Fc) at the electrode is developed. The ECL intensity is correlated to the distance which is controlled by the conformation of the ferrocene-labeled DNA molecular beacon (Fc-MB) between the Fc and Ru(bpy)32+ immobilized on the electrode. The conformation adjustment is conducted via complementary DNA hybridizing with the bases in the loop of the Fc-MB and changing the temperature of the Fc-MB and the resultant double-stranded DNA (dsDNA). Those events all result in change of the ECL intensity. With such characteristics, the solid-state Ru(bpy)32+-ECL film has the potential to be applied to reagentless DNA ECL biosensors and to calculate thermodynamic parameters of equilibrium constants of MB binding and the stem-loop formation.

Enzymatic amplification detection of DNA based on “molecular beacon” biosensors

We described a novel electrochemical DNA biosensor based on molecular beacon (MB) probe and enzymatic amplification protocol. The MB modified with a thiol at its 5′ end and a biotin at its 3′ end was immobilized on the gold electrode through mixed self-assembly process. Hybridization events between MB and target DNA cause the conformational change of the MB, triggering the attached biotin group on the electrode surface. Following the specific interaction between the conformation-triggered biotin and streptavidin-horseradish peroxidase (HRP), subsequent quantification of DNA was realized by electrochemical detection of enzymatic product in the presence of substrate. The detection limit is obtained as low as 0.1nM. The presented DNA biosensor has good selectivity, being able to differentiate between a complementary target DNA sequence and one containing G–G single-base mismatches.

Pyrene Excimer Signaling Molecular Beacons for Probing Nucleic Acids

Molecular beacon DNA probes, containing 1-4 pyrene monomers on the 5' end and the quencher DABCYL on the 3' end, were engineered and employed for real-time probing of DNA sequences. In the absence of a target sequence, the multiple-pyrene labeled molecular beacons (MBs) assumed a stem-closed conformation resulting in quenching of the pyrene excimer fluorescence. In the presence of target, the beacons switched to a stem-open conformation, which separated the pyrene label from the quencher molecule and generated an excimer emission signal proportional to the target concentration. Steady-state fluorescence assays resulted in a subnanomolar limit of detection in buffer, whereas time-resolved signaling enabled low-nanomolar target detection in cell-growth media. It was found that the excimer emission intensity could be scaled by increasing the number of pyrene monomers conjugated to the 5' terminal. Each additional pyrene monomer resulted in substantial increases in the excimer emission intensities, quantum yields, and excited-state lifetimes of the hybridized MBs. The long fluorescence lifetime ( approximately 40 ns), large Stokes shift (130 nm), and tunable intensity of the excimer make this multiple-pyrene moiety a useful alternative to traditional fluorophore labeling in nucleic acid probes.

Molecular beacons for isothermal fluorescence enhancement by the cleavage of RNase HII from Chlamydia pneumoniae

This article describes a new assay for isothermal enhancement of fluorescence intensity. The assay is based on the cleavage of duplexes formed by the chimeric DNA–rN 1–DNA molecular beacon (cMB) and target DNA with Chlamydia pneumoniae RNase HII (CpRNase HII). The loop sequence of the cMB, which was designed according to the target sequence, contains a single ribonucleotide. The combination of CpRNase HII cleavage and cMB (RHMB) permitted a 90-fold increase in fluorescence intensity change compared with the hybridization reaction in the presence of the same amount of target DNA. These results indicate that the RHMB assay can enhance the fluorescence signal in real-time monitoring of the target DNA.

Label‐Free and Self‐Signal Amplifying Molecular DNA Sensors Based on Bioconjugated Polyelectrolytes

AbstractHybrid bio/‐synthetic sensory conjugated polyelectrolytes were developed to achieve selective label‐free detection of target oligonucleotides with amplified fluorescence signal in solution. A completely water soluble and highly fluorescent conjugated poly(p‐phenyleneethynylene) (PPE) was rationally designed and synthesized as a signal amplifying unit and chemically modified with carboxylic functional groups at the ends of the polymer chains to bioconjugate with amine functionalized single‐stranded oligonucleotides as a receptor using carbodiimide chemistry. This approach allows the functional groups on the polymers to be effectively linked to DNA without any damage to the conjugated π‐system of the polymers. DNA detection results using the PPE‐DNA hybrid system confirmed large signal amplification by means of efficient Förster energy transfer from the energy harvesting PPE to the fluorescent dye attached to the complementary analyte DNA. To realize label‐free detection, we also connected a DNA molecular beacon to the newly developed conjugated polymer as a self‐signaling molecular switch. A DNA detection study by using the resulting PPE‐DNA beacon and single strand analyte DNAs showed not only signal‐amplification properties but also self‐signaling properties.

Application of a Highly Robust and Efficient Fluorescence‐Resonance‐Energy‐Transfer (FRET) System in DNA

AbstractWe report a feasibility study for the application of our newly developed highly efficient and robust fluorescence‐resonance‐energy‐transfer (FRET) system to DNA. A 2′‐oligodeoxynucleotide, 12, equipped with a quinolinone derivative as donor and a (bathophenanthroline)ruthenium(II) complex as acceptor and having a single uridine as potential cleavage site under basic conditions revealed an intensive FRET, which almost vanished after cleavage of the oligonucleotide under basic conditions (Fig. 7). Furthermore, in the arrangement of a molecular beacon (MB) DNA (see 13), a significant decrease of the FRET was observed after hybridization to a target sequence (Fig. 9). Due to the long decay times of the fluorescence of the Ru‐complex, the system allows for highly sensitive time‐gated measurements.

Improved Fluorescent In Situ Hybridization Method for Detection of Bacteria from Activated Sludge and River Water by Using DNA Molecular Beacons and Flow Cytometry

Fluorescent in situ hybridization (FISH) remains a key technique in microbial ecology. Molecular beacons (MBs) are self-reporting probes that have potential advantages over linear probes for FISH. MB-FISH strategies have been described using both DNA-based and peptide nucleic acid (PNA)-based approaches. Although recent reports have suggested that PNA MBs are superior, DNA MBs have some advantages, most notably cost. The data presented here demonstrate that DNA MBs are suitable for at least some FISH applications in complex samples, providing superior discriminatory power compared to that of corresponding linear DNA-FISH probes. The use of DNA MBs for flow cytometric detection of Pseudomonas putida resulted in approximately double the signal-to-noise ratio of standard linear DNA probes when using laboratory-grown cultures and yielded improved discrimination of target cells in spiked environmental samples, without a need for separate washing steps. DNA MBs were also effective for the detection and cell sorting of both spiked and indigenous P. putida from activated sludge and river water samples. The use of DNA MB-FISH presents another increase in sensitivity, allowing the detection of bacteria in environmental samples without the expense of PNA MBs or multilaser flow cytometry.