Fluorescent DNA Probes Research Articles

A star-shaped DNA probe based onstrand displacement for universal and multiplexed fluorometric detection of genetic variations.

A star-shaped fluorescent DNA probe (S-probe) is described that can recognize target DNA and discriminate it from interfering DNA via strand displacement including branch migration and toehold exchange. The artificially designed S-probe does not harm the strand displacement while it allows the fluorescently labelled strand and the quencher-labelled strand to be shared among different S-probes targeting different genetic variations. Generally, multiplexed detection of different MT/WT pairs requires different fluorophore-labelled and quencher-labelled strands. The two labelled oligonucleotides of S-probe have sequences decoupled from the target/interfering DNA sequence, so the same fluorescent and quencher strands can be used for different S-probes that target different sequences. The sensitivity, specificity, and general applicability of the method toward BRCA 41293497 mutation, KRAS G13D mutation and two types of EGFR mutations (T790M and L858R) were experimentally demonstrated. The limit of quantification of the MT concentration is 2nM, and the detection limit of the low abundance of the target sequence is 5% (40nM of MT strand in the background of 760nM of WT strand). The fluorometric assay with excitation/emission wavelengths of 485/582nm was successfully applied to clinical samples spiked with mutant-type and wild-type DNA. The unique structure of the S-probe provides a useful tool for the regulation of the strand displacement reaction. Conceivably, the star-shaped DNA probe can be widely adopted to multiplexed detection of genetic variations and provide novel insights into the regulation of strand displacement processes as utilized in DNA based nanomachines. Graphical abstract A star-shaped fluorescent DNA probe (S-probe) with a detection limit of 2nM was adopted to multiplexed detection of genetic variations via strand displacement including branch migration and toehold exchange.

Multiplex PCR for detection and quantification of GM potato event EH92-527-1 in food

Aim - the elaboration of the protocol for the quantitative detection of genetically modified (GM) potato event EH92-527-1 in the format of duplex real-time polimerase chain reaction (RT-PCR) with TaqMan® PCR technology. Material and methods. The duplex system included two types of specific DNA primers and fluorescent probes: the 1st was for identifying of the event-specific EH92-527-1 DNA, the 2nd was for identifying of the taxon-specific potato gene Stp23. The selection of PCR parameters was carried out by empirical assortment of primers and probes, Mg2+-ions, deoxyribonucleotides and polymerase stabilizing agent, primers annealing temperature and incubation time for the each cycle stage. The results of this research was the optimization of the reaction mixture composition for EH92-527-1 event and Stp23 gene fragment identification in the duplex system: 2.5× RT-PCR buffer in the presence of ROX, primers specific for the GM potato (EH92- f/EH92-r) and target taxon (GРF3/GРR3) in the amount of 250/250 and 100/100 nM, probes - 200 and 200 nM, respectively; bovine serum albumin - 0.04%; MgCl2 - 3.5 mM, deoxynucleoside triphosphates - 0.3 mM, as well as the temperature-time reaction profile (initial denaturation at 95°C - 5 minutes, the next 45 cycles: 95 °C - 20 seconds, 58 °C - 20 seconds, 62 °C - 40 seconds). Conclusion. The method for the quantitative detection of genetically modified (GM) potato event EH92-527-1 in the format of duplex RT-PCR has been elaborated. The linearity, precision, accuracy and limit of the method definition are confirmed with in vitro studies, that results testify to the method's reliability.

Synthesis, photophysical properties, and DNA-binding of novel A-π-D-π-A' two-photon absorption chromophores

Three novel two-photon absorption chromophores (1BI, 2TAZ, and 3PyB), which have trans-1,4-divinylbenzene π-bridge, methoxy donors, and different acceptors, were synthesized. Their structures were elucidated by means of hydrogen-1 nuclear magnetic resonance, carbon-13 nuclear magnetic resonance, Fourier transform infrared spectroscopy, and high-resolution mass spectrometry. Their photophysical properties, including linear absorption, single-photon excited fluorescence, two-photon absorption, and two-photon excited fluorescence, were systematically investigated in various solvents. The structure-property relationships were studied aided with the density functional theory calculations. In addition, the absorption, single- and two-photon fluorescence spectra of 3PyB-ctDNA were examined to evaluate whether 3PyB could be used as a DNA stain. The possible interaction mechanism of 3PyB-ctDNA was also studied by the NaCl titration and the viscosity experiment. The results indicate that 3PyB may serve as a potential DNA fluorescent probe.

The live cell DNA stain SiR-Hoechst induces DNA damage responses and impairs cell cycle progression

SiR-Hoechst (SiR-DNA) is a far-red fluorescent DNA probe being used widely for time-lapse imaging of living cells that is reported to be minimally toxic at concentrations as high as 10–25 µM. However, measuring nuclear import of Cyclin B1, inhibition of mitotic entry, and the induction of γH2AX foci in cultured human cells reveals that SiR-Hoechst induces DNA damage responses and G2 arrest at concentrations well below 1 µM. SiR-Hoechst is useful for live cell imaging, but it should be used with caution and at the lowest practicable concentration.

A standard photomap of ovarian nurse cell chromosomes and inversion polymorphism in Anopheles beklemishevi

BackgroundAnopheles beklemishevi is a member of the Maculipennis group of malaria mosquitoes that has the most northern distribution among other members of the group. Although a cytogenetic map for the larval salivary gland chromosomes of this species has been developed, a high-quality standard cytogenetic photomap that enables genomics and population genetics studies of this mosquito at the adult stage is still lacking.MethodsIn this study, a cytogenetic map for the polytene chromosomes of An. beklemishevi from ovarian nurse cells was developed using high-resolution digital imaging from field collected mosquitoes. PCR-amplified DNA probes for fluorescence in situ hybridization (FISH) were designed based on the genome of An. atroparvus. The DNA probe obtained by microdissection procedures from the breakpoint region was labelled in a DOP-PCR reaction. Population analysis was performed on 371 specimens collected in 18 locations.ResultsWe report the development of a high-quality standard photomap for the polytene chromosomes from ovarian nurse cells of An. beklemishevi. To confirm the suitability of the map for physical mapping, several PCR-amplified probes were mapped to the chromosomes of An. beklemishevi using FISH. In addition, we identified and mapped DNA probes to flanking regions of the breakpoints of two inversions on chromosome X of this species. Inversion polymorphism was determined in 13 geographically distant populations of An. beklemishevi. Four polymorphic inversions were detected. The positions of common chromosomal inversions were indicated on the map.ConclusionsThe study constructed a standard photomap for ovarian nurse cell chromosomes of An. beklemishevi and tested its suitability for physical genome mapping and population studies. Cytogenetic analysis determined inversion polymorphism in natural populations of An. beklemishevi related to this species’ adaptation.

Development of an upconversion fluorescence DNA probe for the detection of acetamiprid by magnetic nanoparticles separation

An upconversion fluorescence DNA probe which consists of aptamer-conjugated magnet nanoparticles (apt-MNPs) and complementary DNA-conjugated upconversion nanoparticles (cDNA-UCNPs) was developed to detect acetamiprid. Acetamiprid can specifically conjugate with the apt-MNPs to dissociate the cDNA-UCNPs from the apt-MNPs and resulted in reduced fluorescence intensity through an external magnet. The change of fluorescence intensity (△I) is positively related to the concentration of acetamiprid, which can be applied for the quantification of acetamiprid. Under optimal conditions, a linear detection range and detection limit are 0.89–114.18 μg/L and 0.65 μg/L, respectively. The probe was successfully used to detect acetamiprid in spiked paddy water, soil, pear, apple, wheat and cucumber. Average recoveries are 78.2%–103.5% with intra-day relative standard deviations (RSDs) of 2.6%–10.9% and inter-day RSDs of 4.3%–10.2%. The amounts of acetamiprid in the authentic paddy water and pear samples detected by the DNA probe are significantly correlated with that detected by high-performance liquid chromatography (HPLC).

Quantifying the Degree of Aggregation from Fluorescent Dye-Conjugated DNA Probe by Single Molecule Photobleaching Technology for the Ultrasensitive Detection of Adenosine.

In this work, we demonstrated a single molecule photobleaching-based strategy for the ultrasensitive detection of adenosine. A modified split aptamer was designed to specifically recognize individual adenosine molecules in solution. The specific binding of dye-labeled short strand DNA probes onto the elongated aptamer strand in the presence of adenosine resulted in a concentration-dependent self-aggregation process. The degree-of-aggregation (DOA) of the short DNA probes on the elongated aptamer strand could then be accurately determined based on the single molecule photobleaching measurement. Through statistically analyzing the DOA under different target concentrations, a well-defined curvilinear relationship between the DOA and target molecule concentration (e.g., adenosine) was established. The limit-of-detection (LOD) is down to 44.5 pM, which is lower than those recently reported results with fluorescence-based analysis. Owing to the high sensitivity and excellent selectivity, the sensing strategy described herein would find broad applications in biomolecule analysis under complicated surroundings.

Microfluidic electrophoretic non-enzymatic kanamycin assay making use of a stirring bar functionalized with gold-labeled aptamer, of a fluorescent DNA probe, and of signal amplification via hybridization chain reaction.

The authors describe an enzyme-free aptamer-based assay for the determination of the model antibiotic kanamycin (Kana). The method is making use of (a) microfluidic chip electrophoresis; (b) a stirring bar carrying a gold-labeled aptamer probe, and (c) the hybridization chain reaction (HCR) for signal amplification. Firstly, a stirring bar (length: 1cm; diameter: 0.2mm) was modified with a large amount of duplex DNA and then hybridized with aptamer and its partially complementary chains (cDNA). In the presence of Kana, the binding between the Kana and aptamer unwinds the duplex structures and releases a corresponding amount of cDNA into the supernatant. The released cDNA triggers the HCR in the presence of H1 and H2 DNA hairpin to produce a large amount of duplex DNA chains with different lengths. At the same time, the amounts of H1 and H2 are reduced. The decreased signal of H1/H2 after several HCR cycles can be used to quantify kana in the 1pg·mL-1 to 10ng·mL-1, with a detection limit of 0.29pg·mL-1. The signal is generated by reading the fluorescence, best at excitation/emission maxima of 470/525nm. The whole detection process takes 3min only. The assay was employed to the detection of Kana in spiked milk and fish samples. Results are consistent with those of an enzyme linked immunosorbent assay. The assay has high throughput, high selectivity, and high amplification capability. Graphical abstract Schematic of a stirring bar functionalized with gold-labeled aptamer acting as the capture probe. It can capture the target and release primer simultaneously. The primer triggers the hybridization chain reaction inducing the consumption of H1 and H2. After a certain reaction time, the mixture is injected into the MCE platform for microfluidic electrophoretic separation and fluorometric detection.

Nanostructure and Corresponding Quenching Efficiency of Fluorescent DNA Probes.

Based on the fluorescence resonance energy transfer (FRET) mechanism, fluorescent DNA probes were prepared with a novel DNA hairpin template method, with SiO2 coated CdTe (CdTe/SiO2) core/shell nanoparticles used as the fluorescence energy donors and gold (Au) nanoparticles (AuNPs) as the energy acceptors. The nanostructure and energy donor/acceptor ratio in a probe were controlled with this method. The relationship between the nanostructure of the probes and FRET efficiency (quenching efficiency) were investigated. The results indicated that when the donor/acceptor ratios were 2:1, 1:1, and 1:2; the corresponding FRET efficiencies were about 33.6%, 57.5%, and 74.2%, respectively. The detection results indicated that the fluorescent recovery efficiency of the detecting system was linear when the concentration of the target DNA was about 0.0446–2.230 nmol/L. Moreover, the probes showed good sensitivity and stability in different buffer conditions with a low detection limit of about 0.106 nmol/L.

Dibenzopyridoimidazocinnolinium cations: a new family of light-up fluorescent DNA probes

A new family of weakly fluorescent azonia cations with DNA-binding ability by intercalation whose fluorescence intensity increases significantly upon DNA addition is reported. A live-cell staining cells analysis showed the capacity of these new compounds for active uptake and accumulation by living cells.

Studies on interactions of carbazole derivatives with DNA, cell image, and cytotoxicity

DNA-binding agents have been considered as an established opportunity for the development of anticancer drugs and DNA fluorescence probes. This work reported the synthesis of two novel carbazole derivatives (1 and 2) and investigated their DNA binding properties, living cell image, and cytotoxicity. The results demonstrated that both compounds presented the higher binding affinity to G-quadruplex than to duplex DNA by means of UV–Vis absorption titration and fluorescent intercalator displacement. Continuous variation analysis indicated that their binding stoichiometries of the compound/G-quadruplex were near 2 except the compound/bcl-2. Circular dichroism spectra showed that both compounds had no influence on the conformation of G-quadruplexes. Fluorescence titrations indicated that 2 had the potential to be a G-quadruplex selective fluorescent probe, while 1 could be used as a fluorescent probe for duplex DNA. Confocal fluorescence images indicated that both compounds could enter the living HepG2 cells, and 1 mainly located in nucleus whereas 2 mainly distributed in cytoplasm. DNase and RNase digest tests indicated that both compounds could enter into the nucleus and interact with duplex DNA, especially, 2 could interact with RNA and/or G-quadruplex DNA. They also exhibited an obvious antiproliferative activity to HepG2 by using MTT assays, with IC50 values of 2.7 and 9.5 μM for 1 and 2, respectively.

Anticancer activity of new imidazole derivative of 1R,2R-diaminocyclohexane palladium and platinum complexes as DNA fluorescent probes

The aim of this study was synthesis of two new water-soluble fluorescent palladium and platinum complexes with formulas of [Pt(DACH)(FIP)](NO3)2 and [Pd(DACH)(FIP)](NO3)2, respectively, where FIP is 2-(furan-2-yl)-1H-imidazo[4,5-f][1,10] phenanthroline and DACH is 1R,2R-diaminocyclohexane. Fluorescence spectroscopy, circular dichroism (CD), thermal denaturation measurement, ionic strength, and kinetic study displayed groove binding of Pt complex on DNA, while due to binding of Pd complex, B form of DNA convert to Z form. Due to electrostatic interaction of Pd complex with DNA, the DNA form is converted and it provides enough space for Pd complex to insert between base stacking of DNA. UV–vis study shows two complexes could denature the DNA at low concentrations in exothermic process and Pt complex is more active than Pd complex. Finally, the anticancer and growth inhibitory activities of synthesized complexes were investigated against human colon cancer cell line HCT116 after incubation time of 24 h using MTT assay and higher activity was observed for the platinum complex. Interaction of the two metal derivative complexes was studied by molecular docking and molecular dynamics simulation. The results showed that Pt complexes have higher negative docking energy and higher tendency for interaction with DNA, and exert more structural change on DNA.

Automated Solid-Phase Click Synthesis of Oligonucleotide Conjugates: From Small Molecules to Diverse N-Acetylgalactosamine Clusters.

We developed a novel technique for the efficient conjugation of oligonucleotides with various alkyl azides such as fluorescent dyes, biotin, cholesterol, N-acetylgalactosamine (GalNAc), etc. using copper-catalysed alkyne-azide cycloaddition on the solid phase and CuI·P(OEt)3 as a catalyst. Conjugation is carried out in an oligonucleotide synthesizer in fully automated mode and is coupled to oligonucleotide synthesis and on-column deprotection. We also suggest a set of reagents for the construction of diverse conjugates. The sequential double-click procedure using a pentaerythritol-derived tetraazide followed by the addition of a GalNAc or Tris-GalNAc alkyne gives oligonucleotide-GalNAc dendrimer conjugates in good yields with minimal excess of sophisticated alkyne reagents. The approach is suitable for high-throughput synthesis of oligonucleotide conjugates ranging from fluorescent DNA probes to various multi-GalNAc derivatives of 2'-modified siRNA.

Development of a light-up fluorescent probe for HRAS G-quadruplex DNA

The development of small-molecule G-quadruplex DNA probes has attracted significant attention in recent years. However, G-quadruplexes can display a wide variety of topologies, which process different structures and functions. Therefore, selective discrimination one G-quadruplex structure over another is promising. Herein, we reported the design, synthesis and biological evaluation of a long-chain fatty amine functionalized triphenylamine-quinolinium conjugate 1b. Significant enhancement of the fluorescence intensity (over 180 fold) was observed when 1b bound with HRAS G-quadruplex DNA, while much weaker enhancements were presented in the presence of other G-quadruplexes (45–90-fold) and single/double-stranded DNAs (less than 20-fold), indicating 1b had an excellent selectivity to HRAS. The details of the interactions were investigated by UV–Vis, FID and CD analysis. The results show 1b could interact and stabilize HRAS structure mainly by π-π stacking binding mode. The introduced amine chain of the structure core was found to be better in the terms of inducing selectivity toward G-quadruplex structure. In addition, the application of 1b as a fluorescent agent for living cell imaging was also demonstrated.

G-quadruplex DNA fluorescence sensing by a bis-amine-substituted styrylquinolinium dye

Searching for specific G-quadruplex DNA probes is important for study of the function of G-rich gene sequence, as well as design of novel effective anticancer drugs. In this paper, a novel bis-amine-substituted styrylquinolinium dye (BSAQ) was designed and synthesized to enhance the performance for the application as a G-quadruplex DNA probe. The studies on BSAQ with different DNA forms showed that it could be used as a colorimetric and red-emitting fluorescent probe for G-quadruplex DNA. The limits of detection of BASQ with various G-quadruplex DNAs were found to below 1 nM. CD spectroscopy analysis revealed that BSAQ did not induce the G-rich sequence folding into G-quadruplex structure. These results of this study gave some crucial factors on developing of effective probes for G-quadrupex DNA applications.

Rapid Detection of Urinary Tract Infections via Bacterial Nuclease Activity

Rapid and accurate bacterial detection methods are needed for clinical diagnostic, water, and food testing applications. The wide diversity of bacterial nucleases provides a rich source of enzymes that could be exploited as signal amplifying biomarkers to enable rapid, selective detection of bacterial species. With the exception of the use of micrococcal nuclease activity to detect Staphylococcus aureus, rapid methods that detect bacterial pathogens via their nuclease activities have not beendeveloped. Here, we identify endonuclease I as a robust biomarker for E.coli and develop a rapid ultrasensitive assay that detects its activity. Comparison of nuclease activities of wild-type and nuclease-knockout E.coli clones revealed that endonuclease I is the predominant DNase in E.coli lysates. Endonuclease I is detectable by immunoblot and activity assays in uropathogenic E.coli strains. A rapid assay that detects endonuclease I activity in patient urine with an oligonucleotide probe exhibited substantially higher sensitivity for urinary tract infections than that reported for rapid urinalysis methods. The 3hr turnaround time is much shorter than that of culture-based methods, thereby providing a means for expedited administration of appropriate antimicrobial therapy. We suggest this approach could address various unmet needs for rapid detection of E.coli.

Exploring Fluorescent Dyes at Biomimetic Interfaces with Second Harmonic Generation and Molecular Dynamics.

The adsorption of a DNA fluorescent probe belonging to the thiazole orange family at the dodecane/water and dodecane/phospholipid/water interfaces has been investigated using a combination of surface second harmonic generation (SSHG) and all-atomistic molecular dynamics (MD) simulations. Both approaches point to a high affinity of the cationic dye for the dodecane/water interface with a Gibbs free energy of adsorption on the order of -45 kJ/mol. Similar affinity was observed with a monolayer of negatively charged DPPG (1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol)) lipids. On the other hand, no significant adsorption could be found with the zwitterionic DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) lipids. This was rationalized in terms of Coulombic interactions between the monolayer surface and the cationic dye. The similar affinity for the interface with and without DPPG, despite the favorable Coulombic attraction in the latter case, could be explained after investigating the interfacial orientation of the dye. In the absence of a monolayer, the dye adsorbs with its molecular plane almost flat at the interface, whereas in the presence of DPPG it has to intercalate into the monolayer and adopt a significantly different orientation to benefit from the electrostatic stabilization.

Telomerase Activity Detection with Amplification-Free Single Molecule Stochastic Binding Assay.

Because the elongation of telomeres has been associated with tumorigenesis, it is of great interest to develop rapid and high-confidence telomerase activity detection methods for disease diagnosis. Currently, amplification-based strategies have been extensively explored for telomerase detection in vitro and in vivo. However, amplification is typically associated with poor reproducibility and high background, which hamper further applications of the strategies, particularly for real sample assays. Here, we demonstrate a new amplification-free single molecule imaging method for telomerase activity detection in vitro based on nucleic acid stochastic binding with total internal reflection fluorescence microscopy. The dynamic stochastic binding of a short fluorescent DNA probe with a genuine target yields a distinct kinetic signature from the background noise, allowing us to identify telomerase reaction products (TRPs) at the single molecule level. A limit-of-detection as low as 0.5 fM and a dynamic range of 0.5-500 fM for TRP detection were readily achieved. With this method, telomerase extracted from cancer cells was determined with sensitivity down to 10 cells. Moreover, the length distribution of TRPs was also determined by multiple stochastic probing, which could provide deep insight into the mechanistic study of telomerase catalysis.

Comparison of MoS2, WS2, and Graphene Oxide for DNA Adsorption and Sensing.

Interfacing DNA with two-dimensional (2D) materials has been intensely researched for various analytical and biomedical applications. Most of these studies have been performed on graphene oxide (GO) and two metal dichalcogenides, molybdenum disulfide (MoS2) and tungsten disulfide (WS2); all of them can all adsorb single-stranded DNA. However, they use different surface forces for adsorption based on their chemical structures. In this work, fluorescently labeled DNA oligonucleotides were used and their adsorption capacities and kinetics were studied as a function of ionic strength, DNA length, and sequence. Desorption of DNA from these surfaces was also measured. DNA is more easily desorbed from GO by various denaturing agents, whereas surfactants yield more desorption from MoS2 and WS2. Our results are consistent with the fact that DNA can be adsorbed by GO via π-π stacking and hydrogen bonding, and MoS2 and WS2 mainly use van der Waals force for adsorption. Finally, fluorescent DNA probes were adsorbed by these 2D materials for detecting complementary DNA. For this assay, GO gave the highest sensitivity, whereas they all showed a similar detection limit. This study has enhanced our fundamental understanding of DNA adsorption by two important types of 2D materials and is useful for further rational optimization of their analytical and biomedical applications.

PhenDV, a turn-off fluorescent quadruplex DNA probe for improving the sensitivity of drug screening assays.

We report a new turn-off fluorescent probe, PhenDV, for the identification of high affinity quadruplex (G4) DNA ligands. This push-pull fluorophore displays a high fluorescence quantum yield in water (ΦF = 0.21) and is a selective and strong quadruplex DNA binder. We describe its use as a fluorescent indicator for the G4 Fluorescent Intercalator Displacement (FID) assay as its fluorescence is strongly quenched when bound to G4 DNA and fully restored when displaced by ligand. This probe improves the sensitivity of the G4-FID assay, as the read out relies on increased fluorescence instead of quenching observed with classical on/off probes.