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.     

A portable generic DNA bioassay system based on in situ oligonucleotide synthesis and hybridization detection

In this study, we present a portable and generic DNA bioassay system based on in situ oligonucleotide synthesis followed by hybridization based detection. The system include two main parts, an oligonucleotide synthesizer and a fluorescence detection system. The oligonucleotide synthesizer is based on microfluidic technology and capable of synthesizing any desired oligonucleotide which can be either used as a primer for PCR based detection (external) or a probe for hybridization based detection (integrated) of a target DNA analyte. The oligonucleotide sequence can be remotely sent to the system. The integrated fluorescence detection system is based on a photodiode to detect Texas Red fluorophore as low as 0.5 fmol. The complete system, integrating the oligonucleotide synthesizer and fluorescence detection system, was successfully used to distinguish DNA from two different bacteria strains. The presented generic portable instrument has the potential to detect any desired DNA target sequence in the field. Potential applications are for homeland security and fast responses to emerging bio-threats.     

Steady-state fluorescence emission from the fluorescent probe, 5-iodoacetamidofluorescein, bound to hemoglobin

In the past, fluorescence emission from an extrinsic fluorophore bound to heme-proteins would only be studied with the removal of the heme since fluorescence from the fluorophore could not be detected using right-angle optics. Using front-face fluorometry, a significant steady state emission signal originating from the probe bound to hemoglobin is detected. This is the first report of the detection of extrinsic fluorescence of a probe bound to a heme-protein. We also demonstrate that the extrinsic probe, 5-iodoacetamidofluorescein, is covalently bound to hemoglobin, specifically at beta 93 Cysteine. Ligand binding results in a change in the fluorophore fluorescence intensity as predicted by hemoglobin crystallographic studies. Efficiency of energy transfer measurements are made.     

Stem-loop probe with universal reporter for sensing unlabeled nucleic acids

In this article, we present the design principles and application of a motif composed of a stem-loop probe (SP) hybridized to a fluorescently labeled universal reporter (UR) for sensing unlabeled nucleic acids. At room temperature, SP-UR is in the hairpin-closed form in which the fluorophore of UR is in proximity to the G bases of the hairpin, where consequently the fluorescent emission is quenched significantly. On hybridization with target, SP-UR is trapped in the hairpin-opened configuration in which the fluorophore and the G quenchers are apart. This turns off quenching, increases emission intensity, and signals the presence of target. Compared with the common approach that employs an oligonucleotide probe with a covalently linked fluorophore, the use of a fluorescently labeled universal reporter strand hybridized to an unlabeled stem-loop probe provides a more efficient approach to the fabrication of nucleic acid sensors and microarrays potentially useful for real-time analysis.     

Methylcytosine-selective fluorescence quenching by osmium complexation

We report on the control of the emission from a fluorophore fixed on DNA using the methylcytosine-selective addition of an osmium-bipyridine complex. We have synthesized DNA modified by a microenvironment-sensitive fluorophore, 2-dimethylamino-6-acyl-naphthalene. The emission from the fluorophore tethered to a probe DNA was effectively quenched by a methylcytosine glycol-osmium-bipyridine triad, which was located in the immediate neighborhood of the fluorophore. The discrimination of the cytosine methylation status at a methylation hot spot in the p53 gene was also executed using a well-designed fluorescent DNA probe.     

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.     

Fluorescence resonance energy transfer (FRET) using ssDNA binding fluorescent dye

There is a need for simple and inexpensive methods for genotyping single nucleotide polymorphisms (SNPs) and short insertion/deletion variations (InDels). In this work, I demonstrate that a single-stranded DNA (ssDNA) binding dye can be used as a donor fluorophore for fluorescence resonance energy transfer (FRET). The method presented is a homogenous assay in which detection is based on the FRET from the fluorescence of the ssDNA dye bound to the unmodified detection primer to the fluorescent nucleotide analog incorporated into this detection primer during cyclic template directed primer extension reaction. Collection of the FRET emission spectrum with a scanning fluorescence spectrophotometer allows powerful data analysis. The fluorescence emission signal is modified by the optical properties of the assay vessel. This seems to be a completely neglected parameter. By proper selection of the optical properties of the assay plate one can improve the detection of the fluorescence emission signal.     

Characterization of the fluorophore 4-heptadecyl-7-hydroxycoumarin: a probe for the head-group region of lipid bilayers and biological membranes

The fluorophore 4-heptadecyl-7-hydroxycoumarin was used as a probe to study the properties of phospholipid bilayers at the lipid-water interface. To this end, the steady-state fluorescence anisotropy, the differential polarized phase fluorometry, and the emission lifetime of the fluorophore were measured in isotropic viscous medium, in lipid vesicles, and in the membrane of vesicular stomatitis virus. In the isotropic medium (glycerol), the probe showed an increase in the steady-state fluorescence anisotropy with a decrease in temperature, but the emission lifetime was unaffected by the change in temperature. In glycerol, the observed and predicted values for maximum differential tangents of the probe were identical, indicating that in isotropic medium 4-heptadecyl-7-hydroxycoumarin is a free rotator. Nuclear magnetic resonance and differential scanning calorimetric studies with lipid vesicles containing 1-2 mol % of the fluorophore indicated that the packaging density of the choline head groups was affected in the presence of the probe with almost no effect on the fatty acyl chains. The fluorophore partitioned equally well in the gel and liquid-crystalline phase of the lipids in the membrane, and the phase transition of the bilayer lipids was reflected in the steady-state fluorescence anisotropy of the probe. The presence of cholesterol in the lipid vesicles had a relatively small effect on the dynamics of lipids in the liquid-crystalline state, but a significant disordering effect was noted in the gel state. One of the most favorable properties of the probe is that its emission lifetime was unaffected by the physical state of the lipids or by the temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

A single nucleotide polymorphism melt curve assay employing an intercalating dye probe fluorescence resonance energy transfer for forensic analysis

The characterization and use of DNA sequence polymorphisms are an important aspect of forensic analysis. A number of approaches are being explored for single nucleotide polymorphism (SNP) genotyping, but current detection methods are subject to limitations that adversely impact their utility for forensic analysis. We have developed a novel method for genotyping both single and multiple SNPs that uses an intercalating dye and a probe labeled with a single fluorophore to affect a fluorescence energy transfer. Melting curve analysis is then used to distinguish true alleles from mismatched alleles. We term the new method dye probe fluorescence resonance energy transfer (dpFRET). In the current work, development proceeded at first with synthetic DNA template testing to establish proof of concept for the chemistry involved, followed by the design of polymerase chain reaction (PCR)-based genomic DNA assays to demonstrate potential forensic applications. The loci chosen for testing included both nuclear (MHC DRB) and mitochondrial DNA (cytochrome b) genes. A preliminary assessment of the sensitivity limits of the technology indicated that dpFRET was capable of accurately genotyping DNA from one single diploid cell equivalent. This technology could also potentially impact a wide range of nonforensic disciplines to aid in discovery, screening, and association of DNA sequence polymorphisms.     

Styryl-BODIPY based red-emitting fluorescent OFF-ON molecular probe for specific detection of cysteine

We have synthesized a styryl boron-dipyrromethene (BODIPY)/2,4-dinitrobenzenesulfonyl (DNBS) dyad based red-emitting molecular probe for specific detection of cysteine among the biological thiols. The probe shows intensive absorption at 556 nm and the probe is non-fluorescent. The DNBS moiety can be cleaved off by thiols, the red emission of the BODIPY fluorophore at 590 nm is switched on, with an emission enhancement of 46-fold. The probe shows good specificity toward cysteine over other biological molecules, such as glutathione and amino acids. The emission of the probe is pH-independent in the physiological pH range. The probe is used for fluorescent imaging of cellular thiols. Theoretical calculations based on density functional theory (DFT) were used to elucidate the fluorescence sensing mechanism of the probe, which indicate a dark excited state (S(1)) for the probe but an emissive excited state (S(1)) for the cleaved product (i.e. the fluorophore).     

Development of a multiple internal control for clinical diagnostic real-time amplification assays

A major pitfall in most published genomic amplification methods for the detection and identification of human pathogens is that they do not include an internal amplification control in order to achieve an acceptable level of confidence for the absence of false-negative results. By applying composite primer technology, a single multiple internal amplification control DNA molecule was constructed to detect and quantify the hepatitis B virus, human polyomavirus, Epstein-Barr virus, Toxoplasma gondii and cytomegalovirus using real-time PCR. The multiple internal amplification control contains all forward and reverse primer binding regions targeted in the five distinct duplex PCRs, but with a unique probe hybridization site. Multiple internal amplification control detection sensitivity, assessed by Probit analysis, was 58 copies per PCR, associated with an extremely wide dynamic range (8 log(10) units). Moreover, in testing 614 patient samples, PCR inhibition occurred at a frequency of 0-8.8%. Similar multiple internal amplification controls for quantitative PCR-based assays could be designed to accommodate any infectious profiles in a particular institution as they are easy to make and inexpensive.     

Sequential ordering among multicolor fluorophores for protein labeling facility via aggregation-elimination based β-lactam probes

Development of protein labeling techniques with small molecules is enthralling because this method brings promises for triumph over the limitations of fluorescent proteins in live cell imaging. This technology deals with the functionalization of proteins with small molecules and is anticipated to facilitate the expansion of various protein assay methods. A new straightforward aggregation and elimination-based technique for a protein labeling system has been developed with a versatile emissive range of fluorophores. These fluorophores have been applied to show their efficiency for protein labeling by exploiting the same basic principle. A genetically modified version of class A type β-lactamase has been used as the tag protein (BL-tag). The strength of the aggregation interaction between a fluorophore and a quencher plays a governing role in the elimination step of the quencher from the probes, which ultimately controls the swiftness of the protein labeling strategy. Modulation in the elimination process can be accomplished by the variation in the nature of the fluorophore. This diversity facilitates the study of the competitive binding order among the synthesized probes toward the BL-tag labeling method. An aggregation and elimination-based BL-tag technique has been explored to develop an order of color labeling from the equimolar mixture of the labeling probe in solutions. The qualitative and quantitative determination of ordering within the probes toward labeling studies has been executed through SDS-PAGE and time-dependent fluorescence intensity enhancement measurements, respectively. The desirable multiple-wavelength fluorescence labeling probes for the BL-tag technology have been developed and demonstrate broad applicability of this labeling technology to live cell imaging with coumarin and fluorescein derivatives by using confocal microscopy.     

A strand exchange FRET assay for DNA

A new displacement hybridisation method is reported using a single strand DNA probe, labelled with an acceptor fluorophore (oregon green 488). Detection of double stranded sample target is shown, with discrimination between the probe, duplexed during the assay, and free single stranded probe DNA achieved through the FRET from a donor grove fluorophore (Hoechst 33258). A model for the kinetics of the displacement assay is presented and the course of the assay predicted according to probe/target ratios and sequence. The modelled predictions are consistent with the experimental data showing single base pair mismatch discrimination. The pattern of response according to the mismatch/perfect complement ratio in a mixed sample is also considered with an allele-discrimination ratio lying between the homozygous gene and total mismatch case, according to ratio. The assay is shown to be tolerant of different probe concentrations and ratios and through the dual wavelength recorded signals from donor and FRET acceptor, internal baseline correction is achieved with excellent noise reduction through ratiometric measurement.     

Time-resolved detection probe for homogeneous nucleic acid analyses in one-step format

We report here an extension of homogeneous assays based on fluorescence intensity and lifetime measuring on DNA hybridization. A novel decay probe that allows simple one-step nucleic acid detection with subnanomolar sensitivity, and is suitable for closed-tube applications, is introduced. The decay probe uses fluorescence resonance energy transfer (FRET) between a europium chelate donor and an organic fluorophore acceptor. The substantial change in the acceptor emission decay time on hybridization with the target sequence allows the direct separation of the hybridized and unhybridized probe populations in a time-resolved measurement. No additional sample manipulation or self-hybridization of the probes is required. The wavelength and decay time of a decay probe can be adjusted according to the selection of probe length and acceptor fluorophore, thereby making the probes applicable to multiplexed assays. Here we demonstrate the decay probe principle and decay probe-based, one-step, dual DNA assay using celiac disease-related target oligonucleotides (single-nucleotide polymorphisms [SNPs]) as model analytes. Decay probes showed specific response for their complementary DNA target and allowed good signal deconvolution based on simultaneous optical and temporal filtering. This technique potentially could be used to further increase the number of simultaneously detected DNA targets in a simple one-step homogeneous assay.     

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

A novel probe (Smart probe) has been developed for nucleic acid detection. The smart probe is an oligodeoxyribonucleotide carrying a fluorophore and an intercalator internally. Fluorescence of the smart probe is quenched by the intercalator in the absence of target sequence. While upon hybridization the probe emits greater fluorescence due to the interference of quenching by intercalation. The smart probe has been shown to recognize a single base mismatch in the double-stranded form without utilizing thermal stability difference of hybrids.     

Chemical probes shed light on protein function

Site-specific protein labeling with synthetic dyes is an emerging technique for live cell imaging. A protein or peptide tag fused to the protein of interest provides the means for attachment of a fluorophore or other small molecule probe, to allow non-invasive imaging of the dynamics of protein localization. The past two years have seen significant advances in such methods, the publication of a number of new tags for labeling, and the imaginative application of established techniques to tackle previously intractable biological questions.     

FluoroNanogold: an important probe for correlative microscopy

Correlative microscopy is a powerful imaging approach that refers to observing the same exact structures within a specimen by two or more imaging modalities. In biological samples, this typically means examining the same sub-cellular feature with different imaging methods. Correlative microscopy is not restricted to the domains of fluorescence microscopy and electron microscopy; however, currently, most correlative microscopy studies combine these two methods, and in this review, we will focus on the use of fluorescence and electron microscopy. Successful correlative fluorescence and electron microscopy requires probes, or reporter systems, from which useful information can be obtained with each of the imaging modalities employed. The bi-functional immunolabeling reagent, FluoroNanogold, is one such probe that provides robust signals in both fluorescence and electron microscopy. It consists of a gold cluster compound that is visualized by electron microscopy and a covalently attached fluorophore that is visualized by fluorescence microscopy. FluoroNanogold has been an extremely useful labeling reagent in correlative microscopy studies. In this report, we present an overview of research using this unique probe.     

Wavelength-shifting molecular beacons

We describe wavelength-shifting molecular beacons, which are nucleic acid hybridization probes that fluoresce in a variety of different colors, yet are excited by a common monochromatic light source. The twin functions of absorption of energy from the excitation light and emission of that energy in the form of fluorescent light are assigned to two separate fluorophores in the same probe. These probes contain a harvester fluorophore that absorbs strongly in the wavelength range of the monochromatic light source, an emitter fluorophore of the desired emission color, and a nonfluorescent quencher. In the absence of complementary nucleic acid targets, the probes are dark, whereas in the presence of targets, they fluoresce-not in the emission range of the harvester fluorophore that absorbs the light, but rather in the emission range of the emitter fluorophore. This shift in emission spectrum is due to the transfer of the absorbed energy from the harvester fluorophore to the emitter fluorophore by fluorescence resonance energy transfer, and it only takes place in probes that are bound to targets. Wavelength-shifting molecular beacons are substantially brighter than conventional molecular beacons that contain a fluorophore that cannot efficiently absorb energy from the available monochromatic light source. We describe the spectral characteristics of wavelength-shifting molecular beacons, and we demonstrate how their use improves and simplifies multiplex genetic analyses.     

Secretory phospholipase A2 activity in blood serum: The challenge to sense

Excess levels of secretory phospholipase A2 (sPLA2) is known to contribute to several inflammatory diseases including vascular inflammation correlating with coronary events in coronary artery disease. Thus a method to monitor sPLA2 activity in blood serum is urgently needed. Such method is still a challenge since existing fluorescent probes do not allow to monitor sPLA2 activity directly in blood serum. Here we analyze and overcome barriers in sPLA2 sensing methodology and report a fluorescent probe and a kinetic model of its hydrolysis by sPLA2. New probe is designed with a fluorophore and a quencher not interfering binding to the enzyme. At the same time phospholipid matrix bearing the probe promotes efficient initial quenching of the fluorophore. Kinetic model of probe hydrolysis takes into account signal change due to the side processes. The probe and the kinetic model applied together prove the concept that the activity of sPLA can be measured directly in blood serum.