Molecular dynamics of labeled oligonucleotide probes used for the detection of point mutations in DNA.

The fluorescent label BODIPY 576-589 linked to the 5-end of an oligonucleotide via alkyl chain linkers can be used as a probe to detect point mutations in DNA. We have employed fluorescence anisotropy decay and dynamic fluorescence resonance energy transfer (FRET) in order to investigate the molecular origin of the fluorescence lifetime aberrations of BODIPY in the presence of a mismatched base. The results show that both, an increased flexibility of the alcyl chain linker to the BODIPY molecule as well as relaxation of the secondary structure of the whole complex, contribute to the decreased fluorescence lifetimes reported previously.     

Fluorescence stopped-flow studies of single turnover kinetics of E.coli RecBCD helicase-catalyzed DNA unwinding

We have developed and optimized a stopped-flow fluorescence assay for use in studying DNA unwinding catalyzed by Escherichia coli RecBCD helicase. This assay monitors changes in fluorescence resonance energy transfer (FRET) between a pair of fluorescent probes (Cy3 donor and Cy5 acceptor) placed on opposite sides of a nick in duplex DNA. As such, this is an "all-or-none" DNA unwinding assay. Single turnover DNA unwinding experiments were performed using a series of eight fluorescent DNA substrates containing duplex DNA regions ranging from 24 bp to 60 bp. The time-courses obtained by monitoring Cy3 fluorescence display a distinct lag phase that increases with increasing duplex DNA length, reflecting the transient formation of partially unwound DNA intermediates. These Cy3 FRET time-courses are identical with those obtained using a chemical quenched-flow kinetic assay developed previously. The signal from the Cy5 fluorescence probe shows additional effects that appear to specifically monitor the RecD helicase subunit. The continuous nature of this fluorescence assay enabled us to acquire more precise time-courses for many more duplex DNA lengths in a significantly reduced amount of time, compared to quenched-flow methods. Global analysis of the Cy3 and Cy5 FRET time-courses, using an n-step sequential DNA unwinding model, indicates that RecBCD unwinds duplex DNA with an average unwinding rate constant of kU = 200(+/-40) steps s(-1) (mkU = 680(+/-12)bp s(-1)) and an average kinetic step size, m = 3.4 (+/-0.6) bp step(-1) (5 mM ATP, 10 mM MgCl(2), 30 mM NaCl, pH 7.0, 5% (v/v) glycerol, 25.0 degrees C), in excellent agreement with the kinetic parameters determined using quenched-flow techniques. Under these same conditions, the RecBC enzyme unwinds DNA with a very similar rate. These methods will facilitate detailed studies of the mechanisms of DNA unwinding and translocation of the RecBCD and RecBC helicases.     

Distance-dependent photoinduced electron transfer in synthetic single-strand and hairpin DNA

 The singlet state of stilbene-4,4′-dicarboxamide can serve as a fluorescent probe of both DNA conformation and electron transfer. Covalent incorporation of the stilbene-dicarboxamide into DNA structures with restricted conformational mobility results in inhibition of stilbene isomerization and an increase in its fluorescence quantum yield and lifetime. The fluorescence of stilbenedicarboxamide is selectively quenched by proximate guanine, but not by the three other DNA nucleobases. Selective quenching occurs via an electron transfer mechanism in which stilbene serves as the electron acceptor and guanine as the electron donor. Kinetic analysis of the distance dependence of electron transfer in stilbene-bridged hairpins suggests that duplex DNA is more effective than proteins as a medium for electron transfer, but that it does not function as a molecular wire. Received, accepted: 5 January 1998     

Fluorometric properties of the bibenzimidazole derivative hoechst 33258, a fluorescent probe specific for AT concentration in chromosomal DNA

A new fluorescent probe of chromosomal DNA structure in situ, the bibenzimidazole derivative Hoechst 33258, shows enhanced fluorescence with both AT- and GC-rich DNA; however, enhancement by AT-rich DNA is greater than enhancement with GC-rich DNA. When this compound is used as a probe, it produces localized fluorescence which can be correlated with AT concentration in specific chromosome regions. By the use of 33258, Hilwig and Gropp (1972) were able to demonstrate the relatively AT-rich DNA present in centric regions of mouse chromosomes; these regions do not fluoresce brightly when treated with quinacrine because of the presence of guanine residues which are spaced with high periodicity and which therefore efficiently quench quinacrine fluorescence. The data obtained in this study with DNA polymers of defined structure or composition, as test model compounds, suggest that 33258 is a useful cytochemical reagent for generally identifying all types of AT-rich regions in chromosomes, including those which are not demonstrable with quinacrine.     

DFT study of the interaction between the conjugated fluorescein and dabcyl system, using fluorescene quenching method

Molecular beacon is a DNA probe containing a sequence complementary to the target that is flanked by self-complementary termini, and carries a fluorophore and a quencher at the ends. We used the fluorescein and dabcyl as fluorophore and quencher respectively, and studied with DFT calculations at the GGA/DNP level, and taking into account DFT dispersion corrections by the Grimme and Tkatchenko-Scheffler (TS) schemes, the distance, where the most favorable energetic interaction between the fluorophore and quencher in conjugated form occurs. This distance occurs at a separation distance of 29.451?? between the centers of Dabcyl and fluorescein employing the TS DFT dispersion correction scheme, indicating FRET efficiency around 94.28?%. The calculated emission spectra of the conjugated pair in water indicated that the emission and absorption spectrum overlap completely and thus no fluorescence can be observed due to the fluorescence resonance energy transfer (FRET) effect. The DFT results confirmed the experimentally observing fluorescence quenching of the fluorescein-dabcyl conjugated system by FRET.     

Synthesis of N-4-butylamine acridone and its use as fluorescent probe for ctDNA

A novel N-4-butylamine acridone (BAA), which is an acridone derivative was synthesized and characterized by IR, MS and (1)H NMR. The fluorescent characteristics of BAA was investigated in detail and used as the fluorescent probe for detection of calf thymus DNA (ctDNA). It was found that DNA was able to quench the fluorescence of BAA at 426 nm with the excitation at 254 nm. Under optimal conditions, the corresponding linear response range was from 1.0 to 20.0mg/L and the limit of detections (LOD) was 0.020 mg/L (defined as S/N=3). Moreover, the interaction between BAA and ctDNA was investigated by fluorescence, absorption and viscosity measurements. The results suggested that the interaction between BAA and ctDNA is groove binding in nature.     

OFF-to-ON type fluorescent probe for the detection of 8-oxo-dG in DNA by the Adap-masked ODN probe

We have recently reported that Adap (adenosine-1,3-diazaphenoxazine) is an artificial nucleoside analogue for the specific recognition by multiple hydrogen bonding and that its fluorescence is selectively quenched with 8-oxo-2'-deoxyguanosine (8-oxo-dG) in DNA. We now report the development of a new OFF-to-ON type FRET probe, in which one strand contains Adap and another contains natural nucleotides for the formation of a less stable double strand. Each strand was labeled with Cy3 or BHQ2 at the 5'-end or 3'-end, respectively. It was expected in this system that fluorescence of the duplex probe is first quenched by FRET, but the target DNA strand containing 8-oxo-dG at the complementary site of Adap would enhance the displacement reaction of the less stable duplex probe that results in the fluorescence recovery. The results showed that the duplex probe containing the Adap-T base pair exhibited a complete discrimination between 8-oxo-dG and dG in DNA by fluorescence enhancement.     

DNA hybridization detection in a microfluidic channel using two fluorescently labelled nucleic acid probes

A conceptually new technique for fast DNA detection has been developed. Here, we report a fast and sensitive online fluorescence resonance energy transfer (FRET) detection technique for label-free target DNA. This method is based on changes in the FRET signal resulting from the sequence-specific hybridization between two fluorescently labelled nucleic acid probes and target DNA in a PDMS microfluidic channel. Confocal laser-induced microscopy has been used for the detection of fluorescence signal changes. In the present study, DNA hybridizations could be detected without PCR amplification because the sensitivity of confocal laser-induced fluorescence detection is very high. Two probe DNA oligomers (5'-CTGAT TAGAG AGAGAA-TAMRA-3' and 5'-TET-ATGTC TGAGC TGCAGG-3') and target DNA (3'-GACTA ATCTC TCTCT TACAG GCACT ACAGA CTCGA CGTCC-5') were introduced into the channel by a microsyringe pump, and they were efficiently mixed by passing through the alligator teeth-shaped PDMS microfluidic channel. Here, the nucleic acid probes were terminally labelled with the fluorescent dyes, tetrafluororescein (TET) and tetramethyl-6-carboxyrhodamine (TAMRA), respectively. According to our confocal fluorescence measurements, the limit of detection of the target DNA is estimated to be 1.0 x 10(-6) to 1.0 x 10(-7)M. Our result demonstrates that this analytical technique is a promising diagnostic tool that can be applied to the real-time analysis of DNA targets in the solution phase.     

Combination probes with intercalating anchors and proximal fluorophores for DNA and RNA detection

A new class of modified oligonucleotides (combination probes) has been designed and synthesised for use in genetic analysis and RNA detection. Their chemical structure combines an intercalating anchor with a reporter fluorophore on the same thymine nucleobase. The intercalator (thiazole orange or benzothiazole orange) provides an anchor, which upon hybridisation of the probe to its target becomes fluorescent and simultaneously stabilizes the duplex. The anchor is able to communicate via FRET to a proximal reporter dye (e.g. ROX, HEX, ATTO647N, FAM) whose fluorescence signal can be monitored on a range of analytical devices. Direct excitation of the reporter dye provides an alternative signalling mechanism. In both signalling modes, fluorescence in the unhybridised probe is switched off by collisional quenching between adjacent intercalator and reporter dyes. Single nucleotide polymorphisms in DNA and RNA targets are identified by differences in the duplex melting temperature, and the use of short hybridization probes, made possible by the stabilisation provided by the intercalator, enhances mismatch discrimination. Unlike other fluorogenic probe systems, placing the fluorophore and quencher on the same nucleobase facilitates the design of short probes containing multiple modifications. The ability to detect both DNA and RNA sequences suggests applications in cellular imaging and diagnostics.     

Fluorescent quenching-based quantitative detection of specific DNA/RNA using a BODIPY((R)) FL-labeled probe or primer

We have developed a simple method for the quantitative detection of specific DNA or RNA molecules based on the finding that BODIPY((R)) FL fluorescence was quenched by its interaction with a uniquely positioned guanine. This approach makes use of an oligonucleotide probe or primer containing a BODIPY((R)) FL-modified cytosine at its 5'-end. When such a probe was hybridized with a target DNA, its fluorescence was quenched by the guanine in the target, complementary to the modified cytosine, and the quench rate was proportional to the amount of target DNA. This widely applicable technique will be used directly with larger samples or in conjunction with the polymerase chain reaction to quantify small DNA samples.     

Nanoblinker: Brownian Motion Powered Bio-Nanomachine for FRET Detection of Phagocytic Phase of Apoptosis

We describe a new type of bio-nanomachine which runs on thermal noise. The machine is solely powered by the random motion of water molecules in its environment and does not ever require re-fuelling. The construct, which is made of DNA and vaccinia virus topoisomerase protein, can detect DNA damage by employing fluorescence. It uses Brownian motion as a cyclic motor to continually separate and bring together two types of fluorescent hairpins participating in FRET. This bio-molecular oscillator is a fast and specific sensor of 5′OH double-strand DNA breaks present in phagocytic phase of apoptosis. The detection takes 30 s in solution and 3 min in cell suspensions. The phagocytic phase is critical for the effective execution of apoptosis as it ensures complete degradation of the dying cells’ DNA, preventing release of pathological, viral and tumor DNA and self-immunization. The construct can be used as a smart FRET probe in studies of cell death and phagocytosis.     

Blue Fluorescent cGMP Sensor for Multiparameter Fluorescence Imaging

Cyclic GMP (cGMP) regulates many physiological processes by cooperating with the other signaling molecules such as cyclic AMP (cAMP) and Ca²⁺. Genetically encoded sensors for cGMP have been developed based on fluorescence resonance energy transfer (FRET) between fluorescent proteins. However, to analyze the dynamic relationship among these second messengers, combined use of existing sensors in a single cell is inadequate because of the significant spectral overlaps. A single wavelength indicator is an effective alternative to avoid this problem, but color variants of a single fluorescent protein-based biosensor are limited. In this study, to construct a new color fluorescent sensor, we converted the FRET-based sensor into a single wavelength indicator using a dark FRET acceptor. We developed a blue fluorescent cGMP biosensor, which is spectrally compatible with a FRET-based cAMP sensor using cyan and yellow fluorescent proteins (CFP/YFP). We cotransfected them and loaded a red fluorescent probe for Ca²⁺ into cells, and accomplished triple-parameter fluorescence imaging of these cyclic nucleotides and Ca²⁺, confirming the applicability of this combination to individually monitor their dynamics in a single cell. This blue fluorescent sensor and the approach using this FRET pair would be useful for multiparameter fluorescence imaging to understand complex signal transduction networks.     

A new electrochemical biosensor for DNA detection based on molecular recognition and lead sulfide nanoparticles

In this paper, we constructed a new electrochemical biosensor for DNA detection based on a molecule recognition technique. In this sensing protocol, a novel dual-labeled DNA probe (DLP) in a stem–loop structure was employed, which was designed with dabcyl labeled at the 3′ end as a guest molecule, and with a Pb nanoparticle labeled at the 5′ end as electrochemical tag to indicate hybridization. One α-cyclodextrin-modified electrode (α-CD/MCNT/GCE) was used for capturing the DNA hybridization. Initially, the DLP was in the “closed” state in the absence of the target, which shielded dabcyl from the bulky α-CD/MCNT/GCE conjugate due to a steric effect. After hybridization, the loop sequence (16 bases) formed a rigid duplex with the target, breaking the relatively shorter stem duplex (6 bases). Consequently, dabcyl was forced away from the Pb nanoparticle and became accessible by the electrode. Therefore, the target hybridization event can be sensitively transduced via detecting the electrochemical reduction current signal of Pb. Using this method, as low as 7.1 × 10⁻¹⁰ M DNA target had been detected with excellent differentiation ability for even a single mismatch.     

Minor groove binder-conjugated DNA probes for quantitative DNA detection by hybridization-triggered fluorescence

Here we describe the properties of a novel class of oligonucleotide probes capable of sensitive hybridization-triggered fluorescence. These fluorogenic probes, known commercially as MGB Eclipse probes, are characterized by having a conjugated minor groove binder (MGB) ligand at the 5'-end and a fluorophore at the 3'-end. Additionally, they have an efficient quencher moiety at the 5'-end that is useful with a wide variety of fluorescent dyes. Fluorescence of the single-stranded MGB Eclipse probe is efficiently quenched by the interaction of the terminal dye and quencher groups when not hybridized. Upon hybridization to a complementary target, the MGB molecule folds into duplex and hyper-stabilizes it, allowing the use of shorter, more specific probe sequences. The 5'-MGB-quencher group also prevents nuclease digestion by Taq DNA polymerase during PCR. Because of the hybridization-triggered fluorescence and the excellent specificity imparted by the MGB, these 5'-MGB Eclipse probes have great versatility for real-time PCR applications. The high sensitivity and specificity are illustrated using single nucleotide polymorphism detection, viral load determination, and gene expression analysis.     

Molecular beacons for detecting DNA binding proteins

We report here a simple, rapid, homogeneous fluorescence assay, the molecular beacon assay, for the detection and quantification of sequence-specific DNA-binding proteins. The central feature of the assay is the protein-dependent association of two DNA fragments each containing about half of a DNA sequence defining a protein-binding site. Protein-dependent association of DNA fragments can be detected by any proximity-based spectroscopic signal, such as fluorescence resonance energy transfer (FRET) between fluorochromes introduced into these DNA molecules. The assay is fully homogeneous and requires no manipulations aside from mixing of the sample and the test solution. It offers flexibility with respect to the mode of signal detection and the fluorescence probe, and is compatible with multicolor simultaneous detection of several proteins. The assay can be used in research and medical diagnosis and for high-throughput screening of drugs targeted to DNA-binding proteins.     

Conformational dynamics of DNA polymerase probed with a novel fluorescent DNA base analogue

DNA polymerases discriminate between correct and incorrect nucleotide substrates during a "nonchemical" step that precedes phosphodiester bond formation in the enzymatic cycle of nucleotide incorporation. Despite the importance of this process in polymerase fidelity, the precise nature of the molecular events involved remains unknown. Here we report a fluorescence resonance energy transfer (FRET) system that monitors conformational changes of a polymerase-DNA complex during selection and binding of nucleotide substrates. This system utilizes the fluorescent base analogue 1,3-diaza-2-oxophenothiazine (tC) as the FRET donor and Alexa-555 (A555) as the acceptor. The tC donor was incorporated within a model DNA primer/template in place of a normal base, adjacent to the primer 3' terminus, while the A555 acceptor was attached to an engineered cysteine residue (C751) located in the fingers subdomain of the Klenow fragment (KF) polymerase. The FRET efficiency increased significantly following binding of a correct nucleotide substrate to the KF-DNA complex, showing that the fingers had closed over the active site. Fluorescence anisotropy titrations utilizing tC as a reporter indicated that the DNA was more tightly bound by the polymerase under these conditions, consistent with the formation of a closed ternary complex. The rate of the nucleotide-induced conformational transition, measured in stopped-flow FRET experiments, closely matched the rate of correct nucleotide incorporation, measured in rapid quench-flow experiments, indicating that the conformational change was the rate-limiting step in the overall cycle of nucleotide incorporation for the labeled KF-DNA system. Taken together, these results indicate that the FRET system can be used to probe enzyme conformational changes that are linked to the biochemical function of DNA polymerase.     

Design and synthesis of efficient fluorescent dyes for incorporation into DNA backbone and biomolecule detection

We report here the design and synthesis of a series of pi-conjugated fluorescent dyes with D-A-D (D, donor; A, acceptor), D-pi-D, A-pi-A, and D-pi-A for applications as the signaling motif in biological-synthetic hybrid foldamers for DNA detection. The Horner-Wadsworth-Emmons (HWE) reaction and Knoevenagel condensation were demonstrated as the optimum ways for construction of long pi-conjugated systems. Such rodlike chromophores have distinct advantages, as their fluorescence properties are not quenched by the presence of DNA. To be incorporated into the backbone of DNA, the chromophores need to be reasonably soluble in organic solvent for solid-phase synthesis, and therefore a strategy of using flexible tetraethylene glycol (TEG) linkers at either end of these rodlike dyes was developed. The presence of TEG facilitates the protection of the chain-growing hydroxyl group with DMTrCl (dimethoxytrityl chloride) as well as the activation of the coupling step with phosphoramidite chemistry on an automated DNA synthesizer. To form fluorescence resonance energy transfer (FRET) pairs, six synthetic chromophores with blue to red fluorescence have been developed, and those with orthogonal fluorescent emission were chosen for incorporation into DNA-chromophore hybrid foldamers.     

Construction of logic gates with the fluorene-based small molecule/DNA probes

Fluorene-based small molecules (FSMs) have optical properties and can interact with DNA. In this paper, the integrated "INH" and "AND" gates operating in parallel are developed with the fluorene-based small molecule (FSM)/DNA probe. They are activated by taking advantage of the two-step fluorescence resonance energy transfer (FRET) process and the sequence-recognition mechanism of DNA. Then, a "NOT" gate is obtained with a molecular beacon-like probe (FSM-MB) by using the FSM as the fluorophore. Moreover, the "NOT" gate based on the FSM-MB probe can be used as a biosensor and has potential applications in label-free detection of target molecules.     

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.     

Calibration-curve-free quantitative PCR: a quantitative method for specific nucleic acid sequences without using calibration curves

We have developed a simple quantitative method for specific nucleic acid sequences without using calibration curves. This method is based on the combined use of competitive polymerase chain reaction (PCR) and fluorescence quenching. We amplified a gene of interest (target) from DNA samples and an internal standard (competitor) with a sequence-specific fluorescent probe using PCR and measured the fluorescence intensities before and after PCR. The fluorescence of the probe is quenched on hybridization with the target by guanine bases, whereas the fluorescence is not quenched on hybridization with the competitor. Therefore, quench rate (i.e., fluorescence intensity after PCR divided by fluorescence intensity before PCR) is always proportional to the ratio of the target to the competitor. Consequently, we can calculate the ratio from quench rate without using a calibration curve and then calculate the initial copy number of the target from the ratio and the initial copy number of the competitor. We successfully quantified the copy number of a recombinant DNA of genetically modified (GM) soybean and estimated the GM soybean contents. This method will be particularly useful for rapid field tests of the specific gene contamination in samples.