Photophysical properties of fluorescent DNA-dyes bound to single- and double-stranded DNA in aqueous buffered solution.

The absorption and fluorescence spectra, fluorescence quantum yields, lifetimes and time-resolved fluorescence spectra are reported for nine different fluorescent DNA-dyes. The work was initiated in search of a quantitative method to detect the ratio of single-to-double stranded DNA (ssDNA/dsDNA) in solution based on the photophysics of dye-DNA complexes; the result is a comprehensive study providing a vast amount of information for users of DNA strains. The dyes examined were the bisbenzimide or indole-derived stains (Hoechst 33342, Hoechst 33258 and 4',6-diamidino-2-phenylindole), phenanthridinium stains (ethidium bromide and propidium iodide) and cyanine dyes (PicoGreen, YOYO-1 iodide, SYBR Green I and SYBR Gold). All were evaluated under the same experimental conditions in terms of ionic strength, pH and dye-DNA ratio. Among the photophysical properties evaluated only fluorescence lifetimes for the cyanine stilbene dyes allowed a convenient differentiation between ssDNA and dsDNA. The bisbenzimide dyes showed multiexponential decays when bound to either form of DNA, making lifetime-based analysis cumbersome with inherent errors. These dyes also presented biexponential decay when free in aqueous buffered solutions at different pH. A mechanism for their deactivation is proposed based on two different conformers decaying with different kinetics. The phenanthridinium dyes showed monoexponential decays with ssDNA and dsDNA, but there was no discrimination between them. High dye-DNA ratios (e.g. 1:1) resulted in multiexponential decays for cyanine dyes, resulting from energy transfer or self-quenching deactivation. Shifts in both absorption and fluorescence maxima for both ssDNA and dsDNA DNA-cyanine dye complexes were small. Broadening of dye-ssDNA absorption and fluorescence bands for the cyanine dyes relative to dye-dsDNA bands was detected and attributed to higher degrees of rotational freedom in the former.     

Key Structural Elements of Unsymmetrical Cyanine Dyes for Highly Sensitive Fluorescence Turn‐On DNA Probes

Unsymmetrical cyanine dyes, such as thiazole orange, are useful for the detection of nucleic acids with fluorescence because they dramatically enhance the fluorescence upon binding to nucleic acids. Herein, we synthesized a series of unsymmetrical cyanine dyes and evaluated their fluorescence properties. A systematic structure–property relationship study has revealed that the dialkylamino group at the 2‐position of quinoline in a series of unsymmetrical cyanine dyes plays a critical role in the fluorescence enhancement. Four newly designed unsymmetrical cyanine dyes showed negligible intrinsic fluorescence in the free state and strong fluorescence upon binding to double‐stranded DNA (dsDNA) with a quantum yield of 0.53 to 0.90, which is 2 to 3 times higher than previous unsymmetrical cyanine dyes. A detailed analysis of the fluorescence lifetime revealed that the dialkylamino group at the 2‐position of quinoline suppressed nonradiative decay in favor of increased fluorescence quantum yield. Moreover, these newly developed dyes were able to stain the nucleus specifically in fixed HeLa cells examined by using a confocal laser‐scanning microscope.     

Homodimeric monomethine cyanine dyes as fluorescent probes of biopolymers

The fluorescence properties of newly synthesized homodimeric monomethine cyanine dyes in the presence of biopolymers are investigated. They do not fluoresce in TE buffer and bidistilled water but become strongly fluorescent (Q(F)=0.3-0.9) in the region 530-650 nm when bound to dsDNA and ssDNA. The detection limit of dsDNA is about 1.7 ng/ml. Some of dyes studied are able to distinguish between dsDNA and ssDNA, RNA, BSA in solution and gel electrophoresis. The influence of different factors (temperature, pH and viscosity of the medium, presence of histone) on the formation of the dye-biopolymer complexes is investigated. The results of steady-state and dynamic fluorescence measurements concerning the different types of binding between dyes and biopolymers show that the new dyes are applicable in molecular biology as highly sensitive fluorescence labels.     

Fluororeactands for the Detection of Saccharides Based on Hemicyanine Dyes with a Boronic Acid Receptor

Three hemicyanine dyes with boronic acid receptor functions have been synthesized in a two step procedure. These dyes are capable of forming a covalent bond between their boronic acid moiety and the diol moiety of saccharides which causes fluorescence to change. In detail, the indicator dyes exhibit absorbance maxima at around 460 nm and emission at around 600 nm, show increases in fluorescence upon exposure to saccharides and can be used in aqueous solution at physiological pH.     

New unsymmetrical cyanine dyes for real-time thermal cycling

Asymmetric cyanine dyes bind to the minor groove of double stranded DNA (dsDNA) owing to their crescent configuration; therefore, these dyes are widely used as a dsDNA probes. BOXTO-MEE is derived from BOXTO by adding the polar methoxyethoxyethyl tail in order to increase solubility, dissociation rate kinetics, and stability. As a result, BOXTO-MEE showed significant reduction in nonspecific amplification (primer dimers) without significant effect on target sequence amplification, PCR efficiency, and standard curve correlation coefficient. BETIBO is another example of an asymmetric cyanine dye that can binds to dsDNA but is less efficient than BOXTO-MEE for use in real-time PCR. Statistical analysis of reproducibility results shows that BETIBO is not strong enough to be used for quantifying low nucleic acid quantities. Statistical analysis for BOXTO-MEE results shows that there is no significant difference between the efficiency and correlation coefficient achieved by BOXTO-MEE and SYBR Green I, but a significant difference in the dynamic range is observed because BOXTO-MEE has a wider dynamic range. BOXTO-MEE stock solution was stable at −20 °C for more than 1 year and 40 μM solution was stable for 45 days (at least) at 4 °C.     

Focusing and stabilization of bis‐intercalating dye–DNA complexes for high‐sensitive CE‐LIF DNA analysis

Multiple labeling of nucleic acids by intercalative dyes is a promising method for ultrasensitive nucleic acid assays. The properties of the fast dissociation and instability of dye–DNA complexes may prevent from their wide applications in CE‐LIF nucleic acid analysis. Here, we describe an optimum CE focusing method by using appropriately paired sample and separation buffers, Tris‐glycine buffer and Tris‐glycine‐acetic acid buffer. The developed method was applied in both uncoated and polyacrylamide coated fused‐silica capillary‐based CE‐LIF analysis while the sample and separation buffers were conversely used. The complexes of intercalative dye benzoxazolium‐4‐pyridinium dimer and dsDNA were greatly focused (separation efficiency: 1.8 million theoretical plates per meter) by transient isotachophoresis mechanism in uncoated capillary, and moderately focused by transient isotachophoresis in combination of field amplified sample stacking and further stabilized by the paired buffer in polyacrylamide coated capillary. Based on the developed focusing strategy, an ultrasensitive DNA assay was developed for quantitation of calf thymus dsDNA (from 0.02 to 2.14 pM). By the use of an excitation laser power as low as 1 mW, the detection limits of calf thymus dsDNA (3.5 kb) are 7.9 fM in concentration and 2.4×10^?22 mol (150 molecules) in mass. We further demonstrate that the non‐gel sieving CE‐LIF analysis of DNA fragments can be enhanced by the same strategy. Since the presented strategy can be applied to uncoated and coated capillaries and does not require special device, it is also reasonable to extend to the applications in chip‐based CE DNA analysis.     

Highly fluorescent conjugated pyrenes in nucleic acid probes: (phenylethynyl)pyrenecarbonyl-functionalized locked nucleic acids

In recent years, fluorescently labeled oligonucleotides have become a widely used tool in diagnostics, DNA sequencing, and nanotechnology. The recently developed (phenylethynyl)pyrenes are attractive dyes for nucleic acid labeling, with the advantages of long-wave emission relative to the parent pyrene, high fluorescence quantum yields, and the ability to form excimers. Herein, the synthesis of six (phenylethynyl)pyrene-functionalized locked nucleic acid (LNA) monomers M(1)-M(6) and their incorporation into DNA oligomers is described. Multilabeled duplexes display higher thermal stabilities than singly modified analogues. An increase in the number of phenylethynyl substituents attached to the pyrene results in decreased binding affinity towards complementary DNA and RNA and remarkable bathochromic shifts of absorption/emission maxima relative to the parent pyrene fluorochrome. This bathochromic shift leads to the bright fluorescence colors of the probes, which differ drastically from the blue emission of unsubstituted pyrene. The formation of intra- and interstrand excimers was observed for duplexes that have monomers M(1)-M(6) in both complementary strands and in numerous single-stranded probes. If more phenylethynyl groups are inserted, the detected excimer signals become more intense. In addition, (phenylethynyl)pyrenecarbonyl-LNA monomers M(4), M(5), and M(6) proved highly useful for the detection of single mismatches in DNA/RNA targets.     

RNA and DNA association to zwitterionic and charged monolayers at the air-liquid interface

The objective of this work is to establish under which conditions short RNA molecules (similar to miRNA) associate with zwitterionic phospholipids and how this differs from the association with cationic surfactants. We study how the base pairing (i.e., single stranded versus double stranded nucleic acids) and the length of the nucleic acid and the charge of the lipid/surfactant monolayer affect the association behavior. For this purpose, we study the adsorption of nucleic acids to monolayers composed of dipalmitoyl phosphatidylcholine (DPPC) or dioctadecyl-dimethyl-ammoniumbromide (DODAB) using the surface film balance, neutron reflectometry, and fluorescence microscopy. The monolayer studies with the surface film balance suggested that short single-stranded ssRNA associates with liquid expanded zwitterionic phospholipid monolayers, whereas less or no association is detected for double-stranded dsRNA and dsDNA. In order to quantify the interaction and to determine the location of the nucleic acid in the lipid/surfactant monolayer we performed neutron reflectometry measurements. It was shown that ssRNA adsorbs to and penetrates the liquid expanded monolayers, whereas there is no penetration of nucleic acids into the liquid condensed monolayer. No adsorption was detected for dsDNA to zwitterionic monolayers. On the basis of these results, we propose that the association is driven by the hydrophobic interactions between the exposed hydrophobic bases of the ssRNA and the hydrocarbon chains of the phospholipids. The addition of ssRNA also influences domain formation in the DPPC monolayer, leading to fractal-like interconnected domains. The experimental results are discussed in terms of the implication for biological processes and new leads for applications in medicine and biotechnology.     

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     

A cell-permeable green fluorescent probe for dsDNA

The sensitive and selective detection of nucleic acids is important for basic research and many applied fields. Herein, a novel green DEAB-TO-1 was designed and synthesized. DEAB-TO-1 shows significant fluorescence enhancement (800-fold) when bound to ctDNA and a distinct selectivity for dsDNA over RNA (5.9 times). Moreover, the limit of detection is 0.57 ng/mL. It is live-cell-permeant DNA intecalator, indicating a promising candidate for nucleus-specific imaging and a good counter-stain compatibility with other dyes.     

A New Trace Analytic Method for the Determination of Sequence-Specific DNA with Fluorescent Probes

A new method of fluorescence spectrometry detection of single-strand DNA (ssDNA) was established by hybridizing the ssDNA with its complementary ssDNA to form double-stranded DNA (dsDNA). Our results show that the fluorescence intensity increased significantly when the nucleic acid molecular “light switch"(Ru(phen)₂dppx²⁺) or Hoechst 33258 dye interacted with dsDNA, and the fluorescence intensity also increased as the DNA concentration increased. The changing law was also studied about how the fluorescence intensity changed when the two kinds of fluorescent probes interacted with oligonucleotide of different lengths and different sequences, as well as DNA-DNA′ hybridization products. Then, the effect of the bases mismatch, varying length of DNA chain, and different DNA sequences on the fluorescence intensity were explored at the same time, by detecting the specific DNA sequence of avian influenza H1N1 virus, cauliflower mosaic virus, and hepatitis C virus. Additionally, the selectivity, linear range, and sensitivity of the two probes were compared.     

Singlet excitation transfer from poly rA to bound dye at 77 K

The fluorescence of poly rA frozen in an aqueous medium containing sugar is half-quenched by bound dyes at a fractional concentration ≈ 0.01, smaller by an order-of-magnitude than that required to sensitize the room-temperature fluorescence of dyes bound to nucleic acid polymers. There is no quenching of the fluorescence of similarly frozen adenosine solutions having the same absolute concentrations of base and dye.     

Hybridization of DNA and PNA molecular beacons to single-stranded and double-stranded DNA targets

Molecular beacons are sensitive fluorescent probes hybridizing selectively to designated DNA and RNA targets. They have recently become practical tools for quantitative real-time monitoring of single-stranded nucleic acids. Here, we comparatively study the performance of a variety of such probes, stemless and stem-containing DNA and PNA (peptide nucleic acid) beacons, in Tris-buffer solutions containing various concentrations of NaCl and MgCl(2). We demonstrate that different molecular beacons respond differently to the change of salt concentration, which could be attributed to the differences in their backbones and constructions. We have found that the stemless PNA beacon hybridizes rapidly to the complementary oligodeoxynucleotide and is less sensitive than the DNA beacons to the change of salt thus allowing effective detection of nucleic acid targets under various conditions. Though we found stemless DNA beacons improper for diagnostic purposes due to high background fluorescence, we believe that use of these DNA and similar RNA constructs in molecular-biophysical studies may be helpful for analysis of conformational flexibility of single-stranded nucleic acids. With the aid of PNA "openers", molecular beacons were employed for the detection of a chosen target sequence directly in double-stranded DNA (dsDNA). Conditions are found where the stemless PNA beacon strongly discriminates the complementary versus mismatched dsDNA targets. Together with the insensitivity of PNA beacons to the presence of salt and DNA-binding/processing proteins, the latter results demonstrate the potential of these probes as robust tools for recognition of specific sequences within dsDNA without denaturation and deproteinization of duplex DNA.     

Hybridization and reaction-based fluorogenic nucleic acid probes

We developed fluorogenic probes, which are photoactivated in the presence of specific nucleic acid templates with the release of fluorescent dyes. This templated reaction can be used to target specific nucleic acids in complex mixtures. We further demonstrate that this reaction can be monitored by single-molecule fluorescence imaging.     

Rapid Fluorometric Screening of Antibiotics in Seafood

Simple and rapid fluorometric screening methods have been developed based on the competitive binding between the target and an intercalating fluorophore dye to double-stranded-DNA (dsDNA). In this study, the long-wavelength fluorescente dye TOTO-3 was employed as the indicator. Compounds that interact with dsDNA will affect the binding of TOTO-3 to the nucleic acid thereby changing the fluorescence intensity. The analyte concentration is indirectly determined by the decrease in fluorescence intensity. A fiber optic fluorescence screening system was developed for rapid and convenient sample processing. Lambda DNA (48.5 kb) was chosen as a suitable sensing nucleic acid material. Detection of sulfathiazole and chloramphenicol in shrimps using this method was studied in the range of 0.5–25 ng mL⁻¹ of sulfathiazole and of 1–50 ng mL⁻¹ of chloramphenicol. Detection limits of 0.5 ng mL⁻¹ of sulfathiazole and 1 ng mL⁻¹ of chloramphenicol were achieved. This approach is useful as a routine test in the monitoring of antibiotics in the environment or aquaculture products. The easy operation and the rapid and sensitive detection make this a potential high-throughput screening method.     

Synthesis of water-soluble, ring-substituted squaraine dyes and their evaluation as fluorescent probes and labels

A series of ring-substituted squaraines absorbing and emitting in the red and NIR spectral region was synthesized and their spectral and photophysical properties (quantum yields, fluorescence lifetimes) and photostabilities were measured and compared to Cy5, a commonly used fluorescent label. The absorption maxima in aqueous media were found to be between 628 and 667 nm and the emission maxima are between 642 and 685 nm. Squaraine dyes exhibit high extinction coefficients (163,000–265,000 M⁻¹ cm⁻¹) and lower quantum yields (2–7%) in aqueous buffer but high quantum yields (up to 45%) and long fluorescence lifetimes (up to 3.3 ns) in presence of BSA. Dicyanomethylene- and thio-substituted squaraines exhibit an additional absorption around 400 nm with extinction coefficients between 21,500 and 44,500 M⁻¹ cm⁻¹. These dyes are excitable not only with red but also with blue diode lasers or light emitting diodes. Due to the favourable spectral and photophysical properties these dyes can be used as fluorescent probes and labels for intensity- and fluorescence lifetime-based biomedical applications.     

Sequence-specific recognition of HIV-1 DNA based upon nicking-assisted strand displacement amplification and triplex DNA

Nucleic acid amplification test is a reliable method for primary human immunodeficiency virus(HIV) infection diagnosis.Herein, a novel fluorescent method for sequence-specific recognition of DNA fragment of HIV-1 was established based upon nicking-assisted strand displacement amplification(SDA) and triplex DNA. In the presence of target dsDNA, nicking-assisted SDA process generated a lot of ssDNA, which hybridized with molecular beacon to produce signal. The fluorescence intensity was proportional to the concentration of target dsDNA within the range from 5 to 1000 pmol/L, with a detection limit of 1.4 pmol/L. Moreover, it successfully distinguished target dsDNA from the nucleic acid extractive of human blood. Thus this method has the merit of high sensitivity, and it is suitable for sequence-specific recognition of target dsDNA in complex matrices, which made it a potential application in diagnosis of acquired immunodeficiency syndrome(AIDS) in the future.     

ECHO probes: a concept of fluorescence control for practical 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. This critical review points out the recent advances in exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probes, which have a fluorescence-labeled nucleotide in which two molecules of thiazole orange or its derivatives are linked covalently. ECHO probes show absorption shift and emission switching depending on hybridization with the target nucleic acid. The hybridization-sensitive fluorescence emission of ECHO probes and the further modification of probes have made possible a variety of practical applications, such as multicolor RNA imaging in living cells and facile detection of gene polymorphism (144 references).     

Fluorescence study of DNA binding and bending by EcoRI DNA methyltransferase

We have applied fluorescence anisotropy and fluorescence resonance energy transfer (FRET) techniques to study the interaction between EcoRI DNA methyltransferase (M.EcoRI) and its target DNA in solution. Upon binding with M.EcoRI, the dsDNA containing GAATTC bends to flip out the second adenine for methylation. The binding affinity of M.EcoRI to two dsDNA fragments (20 and 38 bp) was studied with fluorescence anisotropy. Their binding constants at different temperatures from 20 to 40 degrees C were obtained, and the thermodynamic parameters of binding were derived. The results showed that M.EcoRI had a higher binding affinity to the short dsDNA strand than to the long one, and its binding to DNA was primarily entropy-driven. By labeling the 5' ends of the 20-bp dsDNA with two fluorescent dyes, fluorescein (FAM) and tetramethylrhodamine (TMR), we were able to monitor the enhanced TMR fluorescence in the presence of M.EcoRI. The end-to-end distance of the dsDNA determined from the FRET efficiency was changed from 72.4 to 63.4 A, and the DNA bending angle was estimated as 57.8 degrees .     

Label-free aptamer-based sensors for L-argininamide by using nucleic acid minor groove binding dyes

The nucleic acid minor groove binding dyes, DAPI and Hoechst 33258, were for the first time used in label-free aptamer-based sensors for L-argininamide. The synergy binding effect results in the enhancement of fluorescence of dyes. The method for detection of L-argininamide is simple, rapid and cost-effective.