Molecular beacon DNA probes and their bioanalytical applications.
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Animals↗
Biomedical subjects
Weihong Tan
Publications and source records attributed to Weihong Tan.
Real-time monitoring of nucleic acid ligation in homogenous solutions using molecular beacons.
Nucleic acids ligation is a vital process in the repair, replication and recombination of nucleic acids. Traditionally, it is assayed by denatured gel electrophoresis and autoradiography, which are not sensitive, and are complex and discontinuous. Here we report a new approach for ligation monitoring using molecular beacon DNA probes. The molecular beacon, designed in such a way that its sequence is complementary with the product of the ligation process, is used to monitor the nucleic acid ligation in a homogeneous solution and in real-time. Our method is fast and simple. We are able to study nucleic acids ligation kinetics conveniently and to determine the activity of DNA ligase accurately. We have studied different factors that influence DNA ligation catalyzed by T4 DNA ligase. The major advantages of our method are its ultrasensitivity, excellent specificity, convenience and real-time monitoring in homogeneous solution. This method will be widely useful for studying nucleic acids ligation process and other nucleic acid interactions.
DNA Ligases↗
Monitoring molecular beacon DNA probe hybridization at the single-molecule level.
We have monitored the reaction dynamics of the DNA hybridization process on a liquid/solid interface at the single-molecule level by using a hairpin-type molecular beacon DNA probe. Fluorescence images of single DNA probes were recorded by using total internal reflection fluorescence microscopy. The fluorescence signal of single DNA probes during the hybridization to individual complementary DNA probes was monitored over time. Among 400 molecular beacon DNA probes that we tracked, 349 molecular beacons (87.5 %) were hybridized quickly and showed an abrupt fluorescence increase, while 51 probes (12.5 %) reacted slowly, resulting in a gradual fluorescence increase. This ratio stayed about the same when varying the concentrations of cDNA in MB hybridization on the liquid/surface interface. Statistical data of the 51 single-molecule hybridization images showed that there was a multistep hybridization process. Our results also showed that photostability for the dye molecules associated with the double-stranded hybrids was better than that for those with the single-stranded molecular beacon DNA probes. Our results demonstrate the ability to obtain a better understanding of DNA hybridization processes using single-molecule techniques, which will improve biosensor and biochip development where surface-immobilized molecular beacon DNA probes provide unique advantages in signal transduction.
Biosensing Techniques↗
Ultrasensitive DNA detection using highly fluorescent bioconjugated nanoparticles.
Sensitive DNA detection is extremely important in clinical diagnostics, gene therapy, and a variety of biomedical studies. We have developed a novel DNA bioanalysis method with a 0.8 fM (0.8 x 10-15 M) detection limit using a bioconjugated fluorescent nanoparticle-based sandwich assay. An organic dye silica nanoparticle is synthesized using a modified reverse microemulsion. The nanoparticles are highly fluorescent, extremely photostable, and easy for bioconjugation for bioanalysis. They exhibit an excellent signaling ability in the presence of trace amounts of DNA targets. With an effective surface modification, nonspecific binding and nanoparticle aggregation are minimized. In addition, the nanoparticle-based DNA bioanalysis assay can effectively discriminate one-base mismatched DNA sequences. We expect this nanoparticle-based assay to be widely useful in a number of biomedical applications where reproducible, selective, and ultrasensitive gene analysis is critical.
Base Sequence↗
Synthetic DNA aptamers to detect protein molecular variants in a high-throughput fluorescence quenching assay.
Real-time protein detection in homogeneous solutions is necessary in many biotechnology and biomedical studies. The recent development of molecular aptamers, combined with fluorescence techniques, may provide an easy and efficient approach to protein elucidation. This report describes the development of a fluorescence-based assay with synthetic DNA aptamers that can detect and distinguish molecular variants of proteins in biological samples in a high-throughput process. We used an aptamer with high affinity for the B chain of platelet-derived growth factor (PDGF), labeled it with a fluorophore and a quencher at the two termini, and measured fluorescence quenching by PDGF. The specific quenching can be used to detect PDGF at picomolar concentrations even in the presence of serum and other cell-derived proteins in cell culture media. This is the first successful application of a synthetic aptamer for the detection of tumor-related proteins directly from the tumor cells. We also show that three highly related molecular variants of PDGF (AA, AB, and BB dimers) can be distinguished from one another in this single-step assay, which can be readily adapted to a microtiter plate assay for high-throughput analysis. The use of fluorescence quenching as a measure of binding between the DNA probe and the target protein eliminates potential false signals that may arise in traditional fluorescence enhancement assays as a result of degradation of the DNA aptamer by contaminating nucleases in biological specimens. This assay is applicable to proteins that are not naturally DNA binding. The excellent specificity, ultrahigh sensitivity, and simplicity of this one-step assay addresses a growing need for high-throughput methods that detect changes in the expression of gene products and their variants in cell cultures and biological specimens.
Animals↗
Collection of trace amounts of DNA/mRNA molecules using genomagnetic nanocapturers.
The collection and then the separation of rare DNA/mRNA targets with single-base mismatches in a complex matrix is critically important in human disease diagnostics, gene expression studies, and gene profiling. The major result of this work is the development and application of a novel genomagnetic nanocapturer (GMNC) for the collection, separation, and detection of trace amounts of DNA/RNA molecules with one single-base difference. The GMNC is constructed by bioconjugating molecular beacon DNA probes onto magnetic nanoparticle surfaces. We have successfully applied the GMNC in artificial buffer solution samples and in cancer cell samples, both containing different proteins and random DNA sequences. Our method has three distinctly useful features: highly efficient collection of trace amount of DNA/mRNA samples down to femtomolar (10(-15) M) concentrations; excellent ability to differentiate single-base-mismatched DNA/mRNA samples by combining the exceptional specificity of molecular beacons and the separation power of magnetic nanoparticles; and real-time monitoring and confirmation of the collected gene products. The newly developed genomagnetic nanocapturers will be highly useful for the collection of trace amounts of DNA/mRNA targets in a variety of sample sources in forensic, medical, and biotechnological fields.
Actins↗
Bioconjugated nanoparticles for DNA protection from cleavage.
We have developed a novel method to protect DNA from cleavage using bioconjugated nanoparticles. Positively charged amino-modified silica nanoparticles have been directly prepared using water-in-oil microemulsion. Plasmid DNA can be easily enriched onto the positively charged nanoparticle surface, and the DNA strands are well protected from enzymatic cleavage. When incubated with nuclease enzyme for enzymatic cleavage, free plasmid DNA strands are completely cleaved, while those on the nanoparticle surfaces are intact. Our results clearly demonstrate unique properties of nanomaterials when combined with biomolecules. Our simple bionanotechnology will be highly useful in DNA separation, manipulation, and detection, and possibly in genetic engineering and gene therapy, as plasmid DNA can be protected in cellular environments without any change in its property.
Animals↗
A real-time assay for DNA sticky-end pairing using molecular beacons.
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Base Pairing↗
Two-dimensional imaging biosensor for the monitoring of lactate released from brain slices.
Real-time monitoring of lactate release from brain slices has been studied with an optical two-dimensional (2D) imaging biosensor. The 2D biosensor is prepared by direct immobilization of lactate dehydrogenase (LDH) molecules onto a flat silica glass surface through a covalent binding mechanism. The biosensor is able to spatially differentiate lactate concentration variations with conventional optical microscopic spatial resolution. This biosensor has the capability to effectively detect lactate down to a concentration of 100 nM. The 2D biosensor responds uniformly with 2.5% RSD from pixel to pixel. With a 100 ms response time, this 2D biosensor has the capability of monitoring simultaneously many cells in one image. We have studied the impact of KCI on lactate release from brain slices. Clear differences have been observed in lactate release for different regions of the tissue. The real-time determination of the newly released lactate from the mouse brain slices clearly demonstrates the feasibility of monitoring lactate release from living specimens. The 2D biosensor will enable us to study cellular communications and possibly other biological processes that require simultaneous temporal and spatial resolution.
Animals↗
Molecular aptamer beacons for real-time protein recognition.
One of the most pressing problems facing those attempting to understand the regulation of gene expression and translation is the necessity to monitor protein production in a variety of metabolic states. Thus far, there is no easy solution that will either identify or quantitate proteins in real time. Here we introduce a novel protein probe, molecular aptamer beacon (MAB), for real time protein recognition and quantitative analysis. The MAB combines the signal transduction mechanism of molecular beacons and the molecular recognition specificity of aptamers. An MAB based on a thrombin-binding aptamer was prepared as a model to demonstrate the feasibility. Significant fluorescent signal change was observed when MAB was bound to thrombin, which is attributed to a significant conformational change in MAB from a loose random coil to a compact unimolecular quadruplex. The MAB recognizes its target protein with high specificity and high sensitivity (112 picomolar thrombin concentration) in homogeneous solutions. Ratiometric imaging has been conducted with MAB labeled with two fluorophores, which makes it feasible for protein quantitation in living specimen. The unique properties of the MAB will enable the development of a class of protein probes for real time protein tracing in living specimen and for efficient biomedical diagnosis in homogeneous solutions.
Base Sequence↗
A selective optode membrane for histidine based on fluorescence enhancement of meso-meso-linked porphyrin dimer.
A plasticized polymer film, poly(vinyl chloride) (PVC) incorporated with a specific porphyrin dimer, is shown to exhibit significant and analytical usefulness for optical response toward histidine. The porphyrin dimer containing a free-base porphyrin and a covalently linked metalloporphyrin is shown to be weakly fluorescent as a result of the photoinduced intramolecular electron transfer from the inner free-base porphyrin in singlet excited state to a low-spin cobalt(II). The fluorescence enhancement of the membrane by histidine is based on favorable extraction of histidine into the bulk organic membrane and complexation with the inner metallopophyrin moiety and inhibiting the electron transfer process. With the optode membrane described, histidine in a sample solution from 0.0045 to 1.53 mM can be determined. The calibration curve of the optode membrane for histidine shows a good correlation with the mathematically derived formalism and, thus, confirms the theoretically expected behavior. The sensor presented exhibits high selectivity toward histidine over several amino acids and common inorganic anions. The optical selectivity coefficients obtained for histidine over other biologically relevant amino acids and anions are shown to meet the selectivity requirements for the monitoring concentration levels of histidine in biological samples. The selective characteristic of the sensor has been discussed in the view of the coordination chemistry of metalloporphyrin.
Algorithms↗
Molecular beacons: fluorogenic probes for living cell study.
Molecular beacons are a new class of fluorescent probes that can report the presence of specific nucleic acids with high sensitivity and excellent specificity. In addition to their current wide applications in monitoring the progress of polymerase chain reactions, their unique properties make them promising probes for the detection and visualization of target biomolecules in living cells. This article is focused on our recent research in exploring the potential of using molecular beacon for living-cell studies in three important areas: the monitoring of mRNA in living cells, the development of ultrasmall DNA/RNA biosensors, and the novel approach of combining molecular beacon's signal transduction mechanism with aptamer's specificity for real-time protein detection. These applications demonstrate molecular beacon's unique properties in bioanalysis and bioassay development.
Animals↗
Design of a Molecular Beacon DNA Probe with Two Fluorophores This work was partially supported by the Office of Naval Research Young Investigator Award N00014-98-1-0621 and by an NSF Career Award (CHE-9733650). We would like to thank Dr. Richard Hogrefe, Paul Imperial, Kelly Christianson, and the Trilink Oligonucleotide Synthesis group for producing the molecular beacons used in this study.
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Journal Article↗
Photostable luminescent nanoparticles as biological label for cell recognition of system lupus erythematosus patients.
In this article, we report a method for cell recognition of system lupus erythematosus (SLE) patients that uses photostable luminescent nanoparticles as biological labels. The luminescent silica nanoparticles are prepared with a water-in-oil microemulsion (W/O) technique. The silica network is produced by the controlled hydrolysis of tetraethylorthosilicate (TEOS) in water nanodroplets with the initiation of ammonia (NH3.H2O). A luminescent compound, tris(2,2'-bipyridyl)dichlororuthenium(II)hexahydrate [Ru(II)(bpy)3]2+, is doped inside as a luminescent signaling element, and the most appropriate dye concentration for the preparation of the nanoparticles with a size of 28 +/- 4 nm has been determined. The luminescent silica nanoparticles are covalently immobilized with goat anti-human immunoglobulin G (IgG), which can recognize SmIgG+ B lymphocytes. We have used antibody-labeled nanoparticles to recognize target SmIgG+ B lymphocytes isolated from the circulating blood of SLE patients. It has been observed that a bioassay based on fluorescent nanoparticles can identify target cells selectively and efficiently. And fluorescent nanoparticle labels also exhibit high photostability. The experiment results have shown that this cell recognition method was an effective one as further proof of the diagnosis of SLE.
2,2'-Dipyridyl↗
Development of organic dye-doped silica nanoparticles for bioanalysis and biosensors.
The combination of two silica precursors, tetraethylorthosilicate and phenyltriethoxysilane, were utilized to synthesize organic dye-doped silica nanoparticles. The hydrophobic nature of phenyltriethoxysilane keeps the organic dye in the silica matrix, whereas the hydrophilic tetraethylorthosilicate-formed silica allows the resulting nanoparticles to be dispersed in aqueous solutions. Characterization of the nanoparticles showed that they could be synthesized in the nanometer range with high photostability and minimal dye leakage. The silica matrix of the nanoparticles allows different routes of surface biomolecular modification for biosensor and bioanalysis applications. We have shown different applications of the nanoparticles in bioanalysis and in biosensing. Biotin interaction of avidin-coated nanoparticles can be used for the determination of biotinylated bovine serum albumin, and the immobilization of glutamate dehydrogenase on the nanoparticle surfaces enables the nanoparticles to be used as biosensors for glutamate determination.
Biosensing Techniques↗