Interactive Fluorophore and Quencher Pairs for Labeling Fluorescent Nucleic Acid Hybridization Probes

The use of fluorescent nucleic acid hybridization probes that generate a fluorescence signal only when they bind to their target enables real-time monitoring of nucleic acid amplification assays. Real-time nucleic acid amplification assays markedly improves the ability to obtain qualitative and quantitative results. Furthermore, these assays can be carried out in sealed tubes, eliminating carryover contamination. Fluorescent nucleic acid hybridization probes are available in a wide range of different fluorophore and quencher pairs. Multiple hybridization probes, each designed for the detection of a different nucleic acid sequence and each labeled with a differently colored fluorophore, can be added to the same nucleic acid amplification reaction, enabling the development of high-throughput multiplex assays. In order to develop robust, highly sensitive and specific real-time nucleic acid amplification assays it is important to carefully select the fluorophore and quencher labels of hybridization probes. Selection criteria are based on the type of hybridization probe used in the assay, the number of targets to be detected, and the type of apparatus available to perform the assay. This article provides an overview of different aspects of choosing appropriate labels for the different types of fluorescent hybridization probes used with different types of spectrofluorometric thermal cyclers currently available.     

Light-up probes: thiazole orange-conjugated peptide nucleic acid for detection of target nucleic acid in homogeneous solution

We have constructed light-up probes for nucleic acid detection. The light-up probe is a peptide nucleic acid (PNA) oligonucleotide to which the asymmetric cyanine dye thiazole orange (TO) is tethered. It combines the excellent hybridization properties of PNA and the large fluorescence enhancement of TO upon binding to DNA. When the PNA hybridizes to target DNA, the dye binds and becomes fluorescent. Free probes have low fluorescence, which may increase almost 50-fold upon hybridization to complementary nucleic acid. This makes the light-up probes particularly suitable for homogeneous hybridization assays, where separation of the bound and free probe is not necessary. We find that the fluorescence enhancement upon hybridization varies among different probes, which is mainly due to variations in free probe fluorescence. For eight probes studied the fluorescence quantum yield at 25 degrees C in the unbound state ranged from 0.0015 to 0.08 and seemed to depend mainly on the PNA sequence. The binding of the light-up probes to target DNA is highly sequence specific and a single mismatch in a 10-mer target sequence was readily identified.     

Homogeneous genotyping assays for single nucleotide polymorphisms with fluorescence resonance energy transfer detection

A homogeneous detection mechanism based on fluorescence resonance energy transfer (FRET) has been developed for two DNA diagnostic tests. In the template-directed dye-terminator incorporation (TDI) assay, a donor dye-labeled primer is extended by DNA polymerase using allele-specific, acceptor dye-labeled ddNTPs. In the dye-labeled oligonucleotide ligation (DOL) assay, a donor dye-labeled common probe is joined to an allele-specific, acceptor dye-labeled probe by DNA ligase. Once the donor and acceptor dyes become part of a new molecule, intramolecular FRET is observed over background intermolecular FRET. The rise in FRET, therefore, can be used as an index for allele-specific ddNTP incorporation or probe ligation. Real time monitoring of FRET greatly increases the sensitivity and reliability of these assays. Change in FRET can also be measured by end-point reading when appropriate controls are included in the experiment. FRET detection proves to be a robust method in homogeneous DNA diagnostic assays.     

Detection of single nucleotide variations by a hybridization proximity assay based on molecular beacons and luminescence resonance energy transfer

A powerful combination of molecular beacon and luminescence resonance energy transfer technology reveals alterations in nucleic acid structure by as little as a single nucleotide in a novel hybridization proximity assay. The assay measures the length of a single-stranded target when a terbium chelate-labeled molecular beacon hybridizes to one side of the nucleic acid segment to be measured and an acceptor probe carrying a convention fluorophore hybridizes to the opposite end of the target. Using a test sequence shortened incrementally by deleting single nucleotides, this assay reports a nearly linear relationship between sequence length and the distance separating acceptor and donor probes. Consequently, this assay can be used to detect alternative splicing, allele types, rearrangements, insertion, and deletion events by measuring separation distances within a predefined region. Furthermore, the use of terbium chelates in molecular beacons can produce exceptionally high signal-to-background ratios compared to the use of conventional fluorophores. Principles of optimal probe design are investigated experimentally and by computational simulations of plausible molecular beacon folding. Some molecular beacon designs form dimers that reduce their maximal response to target sequences. A simple assay to detect such dimers is reported as a tool to help improve the design of molecular beacons. Optimally designed molecular beacons with terbium chelates and hybridization proximity assays are expected to expand their applications in the analysis and screening of genetic diseases.     

MagiProbe: a novel fluorescence quenching-based oligonucleotide probe carrying a fluorophore and an intercalator

Fluorescence is the favored signaling technology for molecular diagnoses. Fluorescence energy transfer-based methods are powerful homogeneous assay tools. A novel oligonucleotide probe, named MagiProbe, which is simple to use, is described, and information given about the duplex formed with a target. The probe internally has a fluorophore and an intercalator. Its fluorescence is quenched by the intercalator in the absence of a target sequence. On hybridization with a target sequence, the probe emits marked fluorescence due to the interference in quenching by intercalation. Furthermore, MagiProbe hybridized with a single-base mismatched target emits less fluorescence than with a perfect matched target. It therefore can detect a single base difference in a double-stranded form with a target.     

Molecular basis of action of HyBeacon fluorogenic probes: a spectroscopic and molecular dynamics study

HyBeacons, novel DNA probes for ultra-rapid detection of single nucleotide polymorphisms, contain a fluorophore covalently attached via a linker group to an internal nucleotide. As the probe does not require a quencher or self-complementarity to function, this study investigates the molecular-level mechanism underlying the increase of fluorescence intensity on hybridization of HyBeacons with target DNA. Spectroscopic ultraviolet-visible and fluorimetric studies, combined with molecular dynamics simulations, indicate projection of the fluorophore moiety away from the target-probe duplex into aqueous solution, although specific linker-DNA interactions are populated. Based on evidence from this study, we propose that for HyBeacons, the mechanism of increased fluorescence on hybridization is due to disruption of quenching interactions in the single-stranded probe DNA between the fluorophore and nucleobases. Hybridization leads to an extended linker conformation, removing the fluorophore from the immediate vicinity of the DNA bases.     

Time-resolved Förster-resonance-energy-transfer DNA assay on an active CMOS microarray

We present an active oligonucleotide microarray platform for time-resolved F?rster-resonance-energy-transfer (TR-FRET) assays. In these assays, immobilized probe is labeled with a donor fluorophore and analyte target is labeled with a fluorescence quencher. Changes in the fluorescence decay lifetime of the donor are measured to determine the extent of hybridization. In this work, we demonstrate that TR-FRET assays have reduced sensitivity to variances in probe surface density compared with standard fluorescence-based microarray assays. Use of an active array substrate, fabricated in a standard complementary metal-oxide-semiconductor (CMOS) process, provides the additional benefits of reduced system complexity and cost. The array consists of 4096 independent single-photon avalanche diode (SPAD) pixel sites and features on-chip time-to-digital conversion. We demonstrate the functionality of our system by measuring a DNA target concentration series using TR-FRET with semiconductor quantum dot donors.     

Solution-phase detection of polynucleotides using interacting fluorescent labels and competitive hybridization

DNA was assayed in a homogeneous format using DNA probes containing hybridization-sensitive labels. The DNA probes were prepared from complementary DNA strands in which one strand was covalently labeled on the 5'-terminus with fluorescein and the complementary strand was covalently labeled on the 3'-terminus with a quencher of fluorescein emission, either pyrenebutyrate or sulforhodamine 101. Probes prepared in this manner were able to detect unlabeled target DNA by competitive hybridization producing fluorescence signals which increased with increasing target DNA concentration. A single pair of complementary probes detected target DNA at a concentration of approximately 0.1 nM in 10 min or about 10 pM in 20-30 min. Detection of a 4 pM concentration of target DNA was demonstrated in 6 h using multiple probe pairs. The major limiting factors were background fluorescence and hybridization rates. Continuous monitoring of fluorescence during competitive hybridization allowed correction for variable sample backgrounds at probe concentrations down to 20 pM; however, the time required for complete hybridization increased to greater than 1 h at probe concentrations below 0.1 nM. A promising application for this technology is the rapid detection of amplified polynucleotides. Detection of 96,000 target DNA molecules in a 50-microliters sample was demonstrated following in vitro amplification using the polymerase chain reaction technique.     

Detection of hybrid formation between peptide nucleic acids and DNA by fluorescence polarization in the presence of polylysine.

A new method for the detection of PNA/DNA hybrids is presented. In this method, short PNA probes (9-13 mer) are labeled with a fluorescent dye and allowed to hybridize to target DNA molecules. A cationic polyamino acid, such as polylysine, is then added to the reaction mixture, whereupon the DNA molecules bind electrostatically to this polycation. The PNA probes, which are uncharged or may carry only a small charge due to the fluorescent dye, do not bind to polylysine unless hybridized to the negatively charged DNA target. The binding of the labeled PNA/DNA hybrid to the high-molecular-weight polymer leads to a significant change in the rotational correlation time of the fluorophore attached to the PNA. This can be conveniently detected by measuring the fluorescence polarization of the latter. The method is completely homogeneous because no separation of free from bound PNA probe is required. The hybridization and dehybridization reactions can be followed in real time. The method has been applied to the typing of single-nucleotide polymorphisms in PCR products.     

Closed-tube human leukocyte antigen DQA1∗05 genotyping assay based on switchable lanthanide luminescence probes

Genotyping in closed tube is commonly performed using polymerase chain reaction (PCR) amplification and allele-specific oligonucleotide probes using fluorescence resonance energy transfer (FRET). Here we introduce a homogeneous human leukocyte antigen (HLA)–DQA1∗05 end-point PCR assay based on switchable lanthanide luminescence probe technology and a simple dried blood sample preparation. The switchable probe technology is based on two non-luminescent oligonucleotide probes: one carrying a non-luminescent lanthanide chelate and the other carrying a light-absorbing antenna ligand. Hybridization of the probes in adjacent positions to the target DNA leads to the formation of a highly luminescent lanthanide chelate complex by self-assembly of the reporter molecules. Performance of the HLA–DQA1∗05 assay was evaluated by testing blood samples collected on sample collection cards and was prepared by lysing the punched samples (3-mm discs) using alkaline reaction conditions and high temperature. Testing of 147 blood samples yielded 100% correlation to the heterogeneous DELFIA technology-based reference assay. Genotyping requires carefully designed probe sequences able to discriminate matched and mismatched target sequences by hybridization. Furthermore, definite genotype discrimination was achieved because inherently non-luminescent switchable probes together with time-resolved measurement mode led to very low background signal level and, therefore, very high signal differences averaging 54-fold between DQA1∗05 and other alleles.     

Competitive reporter monitored amplification (CMA)--quantification of molecular targets by real time monitoring of competitive reporter hybridization

Background

State of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests.

Methodology and Principal Findings

The described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique.

Conclusions and Significance

The system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution for real time monitoring of binding events of fluorescently labelled ligands to surface immobilized probes. With the model assay for the detection of human immunodeficiency virus type 1 and 2 (HIV 1/2), we have been able to observe the amplification kinetics of five targets simultaneously and accommodate two additional hybridization controls with a simple instrument set-up. The ability to accommodate multiple controls and targets into a single assay and to perform the assay on simple and robust instrumentation is a prerequisite for the development of novel molecular point of care tests.     

A molecular beacon DNA microarray system for rapid detection of E. coli O157:H7 that eliminates the risk of a false negative signal

A DNA hybridization based optical detection platform for the detection of foodborne pathogens has been developed with virtually zero probability of the false negative signal. This portable, low-cost and real-time assaying detection platform utilizes the color changing molecular beacon as a probe for the optical detection of the target sequence. The computer-controlled detection platform exploits the target hybridization induced change of fluorescence color due to the F?rster (fluorescence) resonance energy transfer (FRET) between a pair of spectrally shifted fluorophores conjugated to the opposite ends of a beacon (oligonucleotide probe). Unlike the traditional fluorophore-quencher beacon design, the presence of two fluorescence molecules allows to actively visualize both hybridized and unhybridized states of the beacon. This eliminates false negative signal detection characteristic for the fluorophore-quencher beacon where bleaching of the fluorophore or washout of a beacon is indistinguishable from the absence of the target DNA sequence. In perspective, the two-color design allows also to quantify the concentration of the target DNA in a sample down to < =1 ng/microl. The new design is suitable for simultaneous reliable detection of hundreds of DNA target sequences in one test run using a series of beacons immobilized on a single substrate in a spatial format.     

Novel DNA probes with low background and high hybridization-triggered fluorescence

Novel fluorogenic DNA probes are described. The probes (called Pleiades) have a minor groove binder (MGB) and a fluorophore at the 5′-end and a non-fluorescent quencher at the 3′-end of the DNA sequence. This configuration provides surprisingly low background and high hybridization-triggered fluorescence. Here, we comparatively study the performance of such probes, MGB-Eclipse probes, and molecular beacons. Unlike the other two probe formats, the Pleiades probes have low, temperature-independent background fluorescence and excellent signal-to-background ratios. The probes possess good mismatch discrimination ability and high rates of hybridization. Based on the analysis of fluorescence and absorption spectra we propose a mechanism of action for the Pleiades probes. First, hydrophobic interactions between the quencher and the MGB bring the ends of the probe and, therefore, the fluorophore and the quencher in close proximity. Second, the MGB interacts with the fluorophore and independent of the quencher is able to provide a modest (2–4-fold) quenching effect. Joint action of the MGB and the quencher is the basis for the unique quenching mechanism. The fluorescence is efficiently restored upon binding of the probe to target sequence due to a disruption in the MGB–quencher interaction and concealment of the MGB moiety inside the minor groove.     

Magnetic microparticle-based multiplexed DNA detection with biobarcoded quantum dot probes

We have developed a new analytical method to detect multiple DNA simultaneously based on the biobarcoded CdSe/ZnS quantum dot (QD) and magnetic microparticle (MMP). It was demonstrated by using oligonucleotide sequences of 64 bases associated with human papillomavirus 16 and 18 L1 genes (HPV-16 and HPV-18) as model systems. This analytical system involves three types of probes, a MMP probe and two streptavidin-modified QD probes. The MMPs are functionalized with HPV-16 and HPV-18 captures DNA to form MMP probes. The QDs are conjugated with HPV-16 or HPV-18 probe DNA along with FAM- or Rox-labeled random DNA to form HPV-16 and HPV-18 QD probes, respectively. A one-step hybridization reaction was performed by mixing the MMP probes, HPV-16 and HPV-18 target DNA (T-16 and T-18), HPV-16 and HPV-18 QD probes. Afterwards, the hybrid-conjugated microparticles were separated by a magnet and heated to remove the MMPs. Finally, the detections of T-16 and T-18 were done by measuring fluorescence signals of FAM and Rox, respectively. Under the optimum conditions, the fluorescence intensity exhibited a good linear dependence on target DNA concentration in the range from 8 × 10?11 to 8 × 10?? M. The detection limit of T-16 is up to 7 × 10?11 M (3σ), and that of T-18 is 6 × 10?11 M. Compared with other biobarcode assay methods, the proposed method that QDs were used as the solid support has some advantages including shorter preparation time of QD probes, faster binding kinetics and shorter analytical time. Besides, it is simple and accurate.     

Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes

An important consideration in the design of oligonucleotide probes for homogeneous hybridization assays is the efficiency of energy transfer between the fluorophore and quencher used to label the probes. We have determined the efficiency of energy transfer for a large number of combinations of commonly used fluorophores and quenchers. We have also measured the quenching effect of nucleotides on the fluorescence of each fluorophore. Quenching efficiencies were measured for both the resonance energy transfer and the static modes of quenching. We found that, in addition to their photochemical characteristics, the tendency of the fluorophore and the quencher to bind to each other has a strong influence on quenching efficiency. The availability of these measurements should facilitate the design of oligonucleotide probes that contain interactive fluorophores and quenchers, including competitive hybridization probes, adjacent probes, TaqMan probes and molecular beacons.     

Homogeneous time-resolved fluorescence DNA hybridization assay by DNA-mediated formation of an EDTA-Eu(III)-beta-diketonate ternary complex.

A sensitive homogenous time-resolved fluorescence DNA hybridization assay method based on the formation of an EDTA-Eu(3+)-beta-diketonate ternary complex in the DNA hybrid was developed. The new approach combined the use of two DNA probes whose sequences compose the whole complementary strand to the target DNA, in which one probe was labeled with an EDTA-Eu(3+) complex on the 5'-terminus and the other, labeled with a bidentate beta-diketone on the 3'-terminus. After hybridization of two DNA probes with target DNA, EDTA-Eu(3+) and beta-diketone come close to each other, and an EDTA-Eu(3+)-beta-diketonate ternary complex with a strong and long-lived fluorescence was formed; thus the target DNA was detected sensitively with a detection limit of 6 pM (0.6 fmol per assay) by time-resolved fluorescence measurement. In the absence of the target DNA, due to the poor stability of bidentate beta-diketonate-Eu(3+) complex in very diluted solution, only a small amount of ternary fluorescence complex was formed.     

DNA-probes for the highly sensitive identification of single nucleotide polymorphism using single-molecule spectroscopy

This article presents a new, highly sensitive method for the identification of single nucleotide polymorphisms (SNPs) in homogeneous solutions using fluorescently labeled hairpin-structured oligonucleotides (smart probes) and fluorescence single-molecule spectroscopy. While the hairpin probe is closed, fluorescence intensity is quenched due to close contact between the chromophore and several guanosine residues. Upon hybridization to the respective target SNP sequence, contact is lost and the fluorescence intensity increases significantly. High specificity is achieved by blocking sequences containing mismatch with unlabeled oligonucleotides. Time-resolved single-molecule fluorescence spectroscopy enables the detection of individual smart probes passing a small detection volume. This method leads to a subnanomolar sensitivity for this single nucleotide specific DNA assay technique.     

Stability, specificity and fluorescence brightness of multiply-labeled fluorescent DNA probes.

In this work, we studied the fluorescence and hybridization of multiply-labeled DNA probes which have the hydrophilic fluorophore 1-(straightepsilon-carboxypentynyl)-1'-ethyl- 3,3,3', 3'-tetramethylindocarbocyanine-5,5'-disulfonate (Cy3) attached via either a short or long linker at the C-5 position of deoxyuridine. We describe the effects of labeling density, fluorophore charge and linker length upon five properties of the probe: fluorescence intensity, the change in fluorescence upon duplex formation, the quantum yield of fluorescence (Phif), probe-target stability and specificity. For the hydrophilic dye Cy3, we have demonstrated that the fluorescence intensity andPhifare maximized when labeling every 6th base using the long linker. With a less hydrophilic dye, a labeling density this high could not be achieved without serious quenching of the fluorescence. The target specificity of multiply-labeled DNA probes was just as high as compared to the unmodified control probe, however, a less stable probe-target duplex is formed that exhibits a lower melting temperature. A mechanism that accounts for this destabilization is proposed which is consistent with our data. It involves dye-dye and dye-nucleotide interactions which appear to stabilize a single-stranded conformation of the probe.     

High resolution multicolor fluorescence in situ hybridization using cyanine and fluorescein dyes: Rapid chromosome identification by directly fluorescently labeled alphoid DNA probes

We tested DNA probes directly labeled by fluorescently labeled nucleotides (Cy3-dCTP, Cy5-dCTP, FluorX-dCTP) for high resolution uni- and multicolor detection of human chromosomes and analysis of centromeric DNA organization by in situ hybridization. Alpha-satellite DNA probes specific to chromosomes 1, 2, 3, 4 + 9, 5 + 19, 6, 7, 8, 10, 11, 13 + 21, 14 + 22, 15, 16, 17, 18, 20, 22, X and Y were suitable for the accurate identification of human chromosomes in metaphase and interphase cells. Cy3-labeled probes had several advantages: (1) a high level of fluorescence (5–10 times more compared with fluorescein-labeled probes); (2) a low level of fluorescence in solution, allowing the detection of target chromosomes in situ during hybridization without the washing of slides; and (3) high resistance to photobleaching during prolonged (1-2 h) exposure to strong light, thus allowing the use of a high energy mercury lamp or a long integration time during image acquisition in digital imaging microscopy for the determination of weak signals. For di- and multicolor fluorescence in situ hybridization (FISH), we successfully used different combinations of directly fluorophorated probes with preservation of images by conventional microscopy or by digital imaging microscopy. FluorX and Cy3 dyes allowed the use of cosmid probes for mapping in a one-step hybridization experiment. Cyanine-labeled fluorophorated DNA probes offer additional possibilities for rapid chromosome detection during a simple 15-min FISH procedure, and can be recommended for basic research and clinical studies, utilizing FISH.     

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 40 min at 37°C. The method was successfully applied to multicolor DNA detection and the analysis of telomerase activity in extracts from cancer cells.