Towards a fluorescent molecular switch for nucleic acid biosensing

A novel fluorescent molecular switch for the detection of nucleic acid hybridization has been explored in relation to the development of a structure that would be amenable for operation when immobilized for solid-phase analyses. The structure was prepared by self-assembly, and used Neutravidin as the central multivalent docking molecule, a newly synthesized biotinylated long-chain linker for intercalating dye that was modified with thiazole orange (TO) at one end, and a biotinylated probe oligonucleotide. Self-assembly of the biotinylated components on adjacent Neutravidin binding sites allowed for physical placement of an oligonucleotide probe molecule next to tethered TO. The TO located at the end of the flexible linker chain was available to intercalate, and could report if a duplex structure was formed by a probe–target interaction by means of fluorescence intensity. Subsequently, regeneration of the single-stranded probe was possible without loss of the intercalator to solution. The switch constructs were assembled in solution and subsequently immobilized onto biotin functionalized optical fibers to complete the sensor design. Solution-phase fluorescence lifetime data showed a biexponential behavior for switch constructs, suggesting intercalation as well as a significant secondary binding mode for the immobilized TO. It was found that the secondary binding mechanism for the dye to DNA could be decreased, thus shifting the dye to intercalative binding modes, by adjusting the solution conditions to a pH below the pI of Neutravidin, and by increasing the ionic strength of the buffer. Preliminary work demonstrated that it was possible to achieve up to a fivefold increase in fluorescence intensity on hybridization to the target.     

Tethered thiazole orange intercalating dye for development of fibre-optic nucleic acid biosensors

Single-stranded DNA (ssDNA) oligonucleotide in solution, or that is immobilized onto a surface to create a biosensor, can be used as a selective probe to bind to a complementary single-stranded sequence. Fluorescence enhancement of thiazole orange (TO) occurs when the dye intercalates into double-stranded DNA (dsDNA). TO dye has been covalently attached to probe oligonucleotides (homopolymer and mixed base 10mer and 20mer) through the 5′ terminal phosphate group using polyethylene glycol linker. The tethered TO dye was able to intercalate when dsDNA formed in solution, and also at fused silica surfaces using immobilized ssDNA. The results indicated the potential for development of a self-contained biosensor where the fluorescent label was available as part of the immobilized oligonucleotide probe chemistry. The approach was shown to be able to operate in a reversible manner for multiple cycles of detection of targeted DNA sequences.     

Thiazole Orange Dimers in DNA: Fluorescent Base Substitutions with Hybridization Readout

By using (S)‐2‐amino‐1,3‐propanediol as a linker, thiazole orange (TO) was incorporated in a dimeric form into DNA. The green fluorescence (λ=530 nm) of the intrastrand TO dimer is quenched, whereas the interstrand TO dimer shows a characteristic redshifted orange emission (λ=585 nm). Steady‐state optical spectroscopic methods reveal that the TO dimer fluorescence is independent of the sequential base contexts. Time‐resolved pump–probe measurements and excitation spectra reveal the coexistence of conformations, including mainly stacked TO dimers and partially unstacked ones, which yield exciton and excimer contributions to the fluorescence, respectively. The helicity of the DNA framework distorts the excitonic coupling. In particular, the interstrand TO dimer could be regarded as an excitonically interacting base pair with fluorescence readout for DNA hybridization. Finally, the use of this fluorescent readout was representatively demonstrated in molecular beacons.     

基于荧光探针噻唑橙与核酸适体构象变化的钾离子检测

建立了一种基于核酸适体(Aptamer)构象效应和荧光探针噻唑橙(TO)为荧光分子开关进行钾离子检测的光学方法.室温下钾离子可与Aptamer结合形成G-四面体结构,使双链解链变为四面体结构和单链,从而导致TO荧光强度降低.考察了TO浓度、反应温度及反应时间的影响.在最佳实验条件下,钾离子浓度在1.0×10-6 ～2....     

Brightness through Local Constraint—LNA‐Enhanced FIT Hybridization Probes for In Vivo Ribonucleotide Particle Tracking

Imaging the dynamics of RNA in living cells is usually performed by means of transgenic approaches that require modification of RNA targets and cells. Fluorogenic hybridization probes would also allow the analysis of wild‐type organisms. We developed nuclease‐resistant DNA forced intercalation (FIT) probes that combine the high enhancement of fluorescence upon hybridization with the high brightness required to allow tracking of individual ribonucleotide particles (RNPs). In our design, a single thiazole orange (TO) intercalator dye is linked as a nucleobase surrogate and an adjacent locked nucleic acid (LNA) unit serves to introduce a local constraint. This closes fluorescence decay channels and thereby increases the brightness of the probe–target duplexes. As few as two probes were sufficient to enable the tracking of oskar mRNPs in wild‐type living Drosophila melanogaster oocytes.     

Dual fluorophore PNA FIT-probes - extremely responsive and bright hybridization probes for the sensitive detection of DNA and RNA

Fluorescently labeled oligonucleotides are commonly employed as probes to detect specific DNA or RNA sequences in homogeneous solution. Useful probes should experience strong increases in fluorescent emission upon hybridization with the target. We developed dual labeled peptide nucleic acid probes, which signal the presence of complementary DNA or RNA by up to 450-fold enhancements of fluorescence intensity. This enabled the very sensitive detection of a DNA target (40 pM LOD), which was detectable at less than 0.1% of the beacon concentration. In contrast to existing DNA-based molecular beacons, this PNA-based method does not require a stem sequence to enforce dye-dye communication. Rather, the method relies on the energy transfer between a "smart" thiazole orange (TO) nucleotide, which requires formation of the probe-target complex in order to become fluorescent, and terminally appended acceptor dyes. To improve upon fluorescence responsiveness the energy pathways were dissected. Hydrophobic, spectrally mismatched dye combinations allowed significant (99.97%) decreases of background emission in the absence of a target. By contrast, spectral overlap between TO donor emission and acceptor excitation enabled extremely bright FRET signals. This and the large apparent Stokes shift (82 nm) suggests potential applications in the detection of specific RNA targets in biogenic matrices without the need of sample pre-processing prior to detection.     

Free-probe fluorescence of light-up probes.

The fluorescence enhancement of light-up probes (thiazole orange (TO) conjugated peptide nucleic acids (PNAs)) upon hybridization to target nucleic acid depends on the probe sequence, mainly due to large variations in free-probe fluorescence. Here we study three probes where the fluorescence in free state varies more than 50-fold. We find that this variation is due to a fraction that has TO intramolecularly "back-bound" to the PNA bases. The intramolecular affinity constant for this unimolecular interaction was determined by temperature titrations using absorption spectroscopy, and the fluorescence quantum yields of the probes in back-bound conformation were calculated. The molar ratio of probes in back-bound conformation was 0.70-0.96 at 30 degrees C and 0.40-0.73 at 60 degrees C, and the fluorescence quantum yield in back-bound conformation varied between 0.0020 and 0.077 at 30 degrees C, and 0.00065-0.029 at 60 degrees C. These data show that the variation in free-probe fluorescence depends mainly on the fluorescence quantum yield of the probe in back-bound conformation and to a much lesser extent on the tendency of the probe to adopt the back-bound conformation. With increasing temperature the free-probe fluorescence decreases owing to both reduced degree of back-binding and a decrease of the fluorescence quantum yield in back-bound conformation.     

Exploring base-pair-specific optical properties of the DNA stain thiazole orange

Double-stranded DNA offers multiple binding sites to DNA stains. Measurements of noncovalently bound dye-nucleic acid complexes are, necessarily, measurements of an ensemble of chromophores. Thus, it is difficult to assign fluorescence properties to base-pair-specific binding modes of cyanine dyes or, vice versa, to obtain information about the local environment of cyanines in nucleic acids by using optical spectroscopy. The feasibility to stain DNA and simultaneously probe local perturbations by optical spectroscopy would be a valuable asset to nucleic acid research. So-called FIT probes (forced intercalation probes) were used to pinpoint the location of the DNA stain thiazole orange (TO) in PNADNA duplexes. A detailed analysis of the base-pair dependence of optical properties is provided and enforced binding of TO is compared with "classical" binding of free TO-PRO1. UV-visible absorbance, circular dichroism (CD) and fluorescence spectroscopy, and melting-curve analyses confirmed site-specific TO intercalation. Thiazole orange exhibited base-specific responses that are not observed in noncovalent dye-nucleic acid complexes, such as an extraordinary dependence of the TO extinction coefficient (+/-60 % variation of the averaged epsilon(max) of 57,000 M(-1) cm(-1)) on nearest-neighbor base pairs. TO signals hybridization, as shown by increases in the steady-state fluorescence emission. Studies of TO fluorescence lifetimes in FIT-PNA and in DNADNA and PNADNA complexes highlighted four different fluorescence-decay processes that may be closed or opened in response to matched or single-mismatched hybridization. A very fast decay process (0.04-0.07 ns) and a slow decay process (2.33-3.95 ns) provide reliable monitors of hybridization, and the opening of a fast decay channel (0.22-0.48 ns) that resulted in an attenuation of the fluorescence emission is observed upon the formation of mismatched base pairs.     

Fluorescent properties of oligonucleotide-conjugated thiazole orange probes

The fluorescence properties of thiazole orange, linked via a (1) hydrophobic alkyl or a (2) hydrophilic ethylene glycol chain to the central internucleotidic phosphate group of a pentadeca-2'-deoxyriboadenylate (dA15), are evaluated. Linkage at the phosphate group yields two stereoisomers, S-isomer of the phosphorus chiral center (Sp) and R-isomer of the phosphorus chiral center (Rp); these are studied separately. The character of the linkage chain and the chirality of the internucleotidic phosphate linkage site influence the fluorescent properties of these thiazole orange-oligonucleotide conjugates (TO-probes). Quantum yields of fluorescence (phifl) of between 0.04 and 0.07 were determined for the single-stranded conjugates. The fluorescence yield increased by up to five times upon hybridization with the complementary sequence (d5'[CACT15CAC3']); (phifl values of between 0.06-0.35 were determined for the double-stranded conjugates. The phifl value (0.17) of thiazole orange, 1-(N,N'-trimethylaminopropyl)-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene]-quinolinium iodide (TO-Pro 1) in the presence of the oligonucleotide duplex (TO-Pro 1: dA15.d5'[CACT15CAC3'] (1:1)) is much less than that for some of the hybrids of the conjugates. Our studies, using steady-state and time-resolved fluorescence experiments, show that a number of discrete fluorescent association species between the thiazole orange and the helix are formed. Time-resolved studies on the four double-stranded TO-probes revealed that the fluorescent oligonucleotide-thiazole orange complexes are common, only the distribution of the species varies with the character of the chain and the chirality at the internucleotidic phosphate site. Those TO-probes in which the isomeric structure of the phosphate-chain linkage is Rp, and therefore such that the fluorophore is directed toward the minor groove, have higher phifl values than the Sp isomer. Of the systems studied, thiazole orange linked by an alkyl chain to the internucleotidic phosphate (Rp isomer) has the highest phifl and the greatest fraction of the longest-lived fluorescent thiazole orange species (in the hybrid form).     

Doubly thiazole orange-labeled cytidine for functional expansion of a hybridization-sensitive probe

A 2′-deoxycytidine derivative modified by two thiazole orange dyes has been designed for functional expansion of hybridization-sensitive fluorescence probes. This nucleotide was synthesized by a procedure that included protection of the cytosine amino group by di(n-butyl)formamidine formation, followed by incorporation into DNA. The fluorescence of the synthesized DNA was controlled by excitonic interaction, showing strong fluorescence upon hybridization with the target nucleic acid and effective quenching in a single-stranded state of the probe.     

Effect of oligonucleotide probes substituted by deoxyinosines on the specificity of SNP detection on the DNA microarray

One of the main factors that can affect the quality of microarray results is the microarray hybridization specificity. The key factor that affects hybridization specificity is the design of the probes. In this paper, we described a novel oligonucleotide probe containing deoxyinosines aimed at improving DNA hybridization specificity. We compared different probes to determine the distance between deoxyinosine base and SNPs site and the number of deoxyinosine bases. The new probe sequences contained two set of deoxyinosines (each set had two deoxyinosines), in which the interval between SNP site and each set of deoxyinosines was two bases. The new probes could obtain the highest hybridization specificity. The experimental results showed that probes containing deoxyinosines hybridized effectively to the perfectly matched target and improved the hybridization specificity of DNA microarray. By including a simple washing step after hybridization, these probes could distinguish matched targets from single‐base‐mismatched sequences perfectly. For the probes containing deoxyinosines, the fluorescence intensity of a match sequence was more than eight times stronger than that of a mismatch. However, the intensity ratio was only 1.3 times or less for the probes without deoxyinosines. Finally, using hybridization of the PCR product microarrays, we successfully genotyped SNP of 140 samples using these new labeled probes. Our results show that this is a useful new strategy for modifying oligonucleotide probes for use in DNA microarray analysis.     

Screening of Minimalist Noncanonical Sites in Duplex DNA and RNA Reveals Context and Motif-Selective Binding by Fluorogenic Base Probes

We hypothesize that programmable hybridization to noncanonical nucleic acid motifs may be achieved by macromolecular display of binders to individual noncanonical pairs (NCPs). As each recognition element may individually have weak binding to an NCP, we developed a semi-rational approach to detect low affinity interactions between selected nitrogenous bases and noncanonical sites in duplex DNA and RNA. A set of fluorogenic probes was synthesized by coupling abiotic (triazines, pyrimidines) and native RNA bases to thiazole orange (TO) dye. This probe library was screened against duplex nucleic acid substrates bearing single abasic, single NCP, and tandem NCP sites. Probe engagement with NCP sites was reported by 100–1000× fluorescence enhancement over background. Binding is strongly context-dependent, reflective of both molecular recognition and stability: less stable motifs are more likely to bind a synthetic probe. Further, DNA and RNA substrates exhibit entirely different abasic and single NCP binding profiles. While probe binding in the abasic and single NCP screens was monotonous, much richer binding profiles were observed with the screen of tandem NCP sites in RNA, in part due to increased steric accessibility. In addition to known binding interactions between the triazine melamine (M) and T/U sites, the NCP screens identified new targeting elements for pyrimidine-rich motifs in single NCPs and 2×2 internal bulges. We anticipate that semi-rational approaches of this type will lead to programmable noncanonical hybridization strategies at the macromolecular level.     

一种高效灵敏的DNA荧光探针的合成及应用

设计合成了一种新型噻唑橙二聚体荧光染料Bi-TO3, 采用荧光发射光谱、 圆二色光谱及活细胞荧光成像等方法研究了其与DNA的相互作用. 在10 mmol/L Tris-HCl缓冲液(pH=7.4)中, Bi-TO3的固有荧光极弱, 量子产率小于0.001%; 与小牛胸腺DNA结合后, 其荧光可显著增强约950倍, 但对RNA和蛋白等生物大分子及黏度等环境因素则无明显响应. 紫外吸收光谱及圆二色光谱滴定实验表明, Bi-TO3以小沟结合模式与DNA作用, 且对AT序列有选择性. 实验结果表明, 在缓冲溶液中Bi-TO3的荧光增强信号与低浓度范围的poly(dA-dT)₂仍呈良好的线性关系, 检出限为13.3 ng/mL, 灵敏较度高; 且Bi-TO3可在较低浓度范围(6~12 μmol/L)内应用于活细胞荧光成像.     

Thiazole orange (TO) as a light-switch probe: a combined quantum-mechanical and spectroscopic study

A Density Functional Theory (DFT) study of the absorbance and fluorescence emission characteristics of the cyanine thiazole orange (TO) in solution and when intercalated in DNA was carried out in combination with spectrophotometric and spectrofluorometric experiments under different conditions (temperature, concentration, solvent viscosity). T-jump relaxation kinetics of the TO monomer-dimer conversion enabled the thermodynamic parameters of this process to be evaluated. The overall data collected provided information on the features of the "light-switch" by the fluorescent TO and the comparison between experimental and calculated photo-physical properties allowed us to explain and rationalize both shifts and quenching/enhancing effects on fluorescence due to solvation, dimerisation and intercalation in the DNA.     

Hybridization‐Sensitive On–Off DNA Probe: Application of the Exciton Coupling Effect to Effective Fluorescence Quenching

The design of dyes that emit fluorescence only when they recognize the target molecule, that is, chemistry for the effective quenching of free dyes, must play a significant role in the development of the next generation of functional fluorescent dyes. On the basis of this concept, we designed a doubly fluorescence‐labeled nucleoside. Two thiazole orange dyes were covalently linked to a single nucleotide in a DNA probe. An absorption band at approximately 480 nm appeared strongly when the probe was in a single‐stranded state, whereas an absorption band at approximately 510 nm became predominant when the probe was hybridized with the complementary strand. The shift in the absorption bands shows the existence of an excitonic interaction caused by the formation of an H aggregate between dyes, and as a result, emission from the probe before hybridization was suppressed. Dissociation of aggregates by hybridization with the complementary strand resulted in the disruption of the excitonic interaction and strong emission from the hybrid. This clear change in fluorescence intensity that is dependent on hybridization is useful for visible gene analysis.     

Visual detection of lead(II) using a label-free DNA-based sensor and its immobilization within a monolithic hydrogel

Lead is highly toxic and its detection has attracted a lot of research interests. In recent years, DNA has been used for Pb(2+) recognition and many fluorescent sensors with low to sub-nM detection limits have been reported. These figures of merit were typically measured using a spectrophotometer that can detect nM DNA with a high signal-to-noise ratio. For visual detection, however, μM DNA or dye was required, making it difficult to detect low nM targets. We recently achieved a visual sensitivity of 10 nM Hg(2+) by immobilizing a DNA probe in a hydrogel. This was made possible because the gel was able to actively adsorb Hg(2+). In this work, we aim to test whether this method can be extended to the detection of Pb(2+). First, a new Pb(2+) sensor was designed based on a guanine-rich DNA and DNA binding dyes such as thiazole orange and SYBR Green I. The free DNA showed a detection limit of 8 nM Pb(2+) using 40 nM DNA. For visual detection in solution with 1 μM of the DNA probe, however, ～300 nM Pb(2+) was required. After immobilization in a monolithic polyacrylamide hydrogel, even 20 nM Pb(2+) could be visually detected with a sample volume of 50 mL. Therefore, sensitive detection without signal amplification was achieved. Finally, we demonstrated simultaneous detection of both Hg(2+) and Pb(2+) in the same water sample with shape encoded hydrogel sensors.     

A theoretical and experimental study of two thiazole orange derivatives with single- and double-stranded oligonucleotides, polydeoxyribonucleotides and DNA

The effect of interaction with DNA and oligonucleotides on the photophysical properties of two thiazole orange (TO) derivatives, with different side chains (-(CH2)3-N+(CH3)3 and -(CH2)6-I)) linked to the nitrogen of the quinoline ring of the thiazole orange, is presented here. The first one called TO-PRO1 is a commercially available dye, whereas the second one called TO-MET has been specially synthesized for further covalent binding to oligonucleotides with the aim of being used for specific in situ detection of biomolecular interactions. Both photophysical measurements and molecular calculations have been done to assess their possible mode of interaction with DNA. When dissolved in buffered aqueous solutions both derivatives exhibit very low fluorescence quantum yields of 8 x 10(-5) and 2 x 10(-4), respectively. However, upon binding to double-stranded DNA, large spectroscopic changes result and the quantum yield of fluorescence is enhanced by four orders of magnitude, reaching values up to phi F = 0.2 and 0.3, respectively, as a result of an intercalation mechanism between DNA base pairs. A modulation of the quantum yield is observed as a function of the base sequence. The two derivatives also bind with single-stranded oligonucleotides, but the fluorescence quantum yield is not so great as that when bound to double-stranded samples. Typical fluorescence quantum yields of 7 x 10(-3) to 3 x 10(-2) are observed when the dyes interact with short oligonucleotides, whereas the fluorescence quantum yield remains below 10(-2) when interacting with single-stranded oligonucleotides. This slight but significant quantum-yield increase is interpreted as a folding of the single strand around the dye, which reduces the internal rotation of the two heterocycles around the central methine bridge that links the two moieties of the dye. From these properties, it is proposed to link monomer covalently to oligonucleotides for the subsequent detection of target sequences within cells.     

Excitonic interaction: another photophysical process for fluorescence‐controlled nucleic acid sensing

An excitonic interaction caused by the H‐aggregation of fluorescent dyes is a new type of useful photophysical process for fluorescence‐controlled nucleic acid sensing. We designed a fluorescence‐labeled nucleotide in which two thiazole orange dyes were linked covalently. A DNA strand containing this fluorescence‐labeled nucleotide showed absorption at 480 nm before hybridization, whereas an absorption band at 510 nm became predominant when the DNA was hybridized with the complementary strand. The shift in the absorption bands shows the existence of an excitonic interaction between dyes in the nucleotide, and as a result, emission from the doubly thiazole orange‐labeled DNA was well controlled. This clear change in fluorescence intensity depending on hybridization is applicable to multicolor RNA imaging in living cells. © 2010 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 10: 188–196; 2010: Published online in Wiley InterScience ( www.interscience.wiley.com ) DOI 10.1002/tcr.201000003     

Ensemble hybridisation--a new method for exploring sequence dependent fluorescence of dye-nucleic acid conjugates

The fluorescence of thiazole orange as artificial base in PNA was investigated in a nearest neighbour analysis; library-to-library hybridisation allowed the identification of probe sequences suitable for homogeneous DNA detection.     

Homogeneous DNA Detection Based on Fluorescence Quenching by Nanoparticles in Single-step Format :Target-Induced Configuration Transform

A new strategy for homogeneous detection of DNA hybridization in single-step format was developed based on fluorescence quenching by gold nanoparticles. The gold nanoparticle is functionalized with 5’-thiolated 48-base oligonucleotide (probe sequence), whose 3’-terminus is labeled with fluorescein (FAM), a negatively charged fluorescence dye. The oligonucleotide adopts an extended configuration due to the electrostatic repulsion between negatively charged gold nanoparticle and the FAM-attached probe sequence. After addition of the complementary target sequence, specific DNA hybridization induces a conformation change of the probe from an extended structure to an arch-like configuration, which brings the fluorophore and the gold nanoparticle in close proximity. The fluorescence is efficiently quenched by gold nanoparticles. The fluorescence quenching efficiency is related to the target concentration, which allows the quantitative detection for target sequence in a sample. A linear detection range from 1.6 to 209.4 nmol/L was obtained under the optimized experimental conditions with a detection limit of 0.1 nmol/L. In the assay system, the gold nanoparticles act as both nanoscaffolds and nanoquenchers. Furthermore, the proposed strategy, in which only two DNA sequences are involved, is not only different from the traditional molecular beacons or reverse molecular beacons but also different from the commonly used sandwich hybridization methods. In addition, the DNA hybridization detection was achieved in homogenous solution in a single-step format, which allows real-time detection and quantification with other advantages such as easy operation and elimination of washing steps.