A homogeneous DNA hybridization system by using a new luminescence terbium chelate

Homogeneous DNA hybridization assay based on the luminescence resonance energy transfer (LRET) from a new luminescence terbium chelate, N,N,N(1),N(1)-[2,6-bis(3'-aminomethyl-1'-pyrazolyl)-4-phenylpyridine]tetrakis(acetic acid) (BPTA)-Tb(3+) (lambda(ex) = 325 nm and lambda(em) = 545 nm) to an organic dye, Cy3 (lambda(ex) = 548 nm and lambda(em) = 565 nm), has been developed. In the system, two DNA probes whose sequences are complementary to the two different consecutive sequences of a target DNA are used; one of the probes is labeled with the Tb(3+) chelate at the 3'-end, and the other is with Cy3 at the 5'-end. Labeling of the Tb(3+) chelate is accomplished via the linkage of a biotin-labeled DNA probe with the Tb(3+) chelate-labeled streptavidin. Strong sensitized emission of Cy3 was observed upon excitation of the Tb(3+) chelate at 325 nm, when the two probe DNAs were hybridized with the target DNA. The sensitivity of the assay was very high compared with those of the previous homogeneous-format assays using the conventional organic dyes; the detection limit of the present assay is about 30 pM of the target DNA strand.     

Homogeneous time-resolved fluorescence DNA hybridization assay by DNA-mediated formation of an EDTA-Eu(III)-beta-diketonate ternary complex.

A sensitive homogenous time-resolved fluorescence DNA hybridization assay method based on the formation of an EDTA-Eu(3+)-beta-diketonate ternary complex in the DNA hybrid was developed. The new approach combined the use of two DNA probes whose sequences compose the whole complementary strand to the target DNA, in which one probe was labeled with an EDTA-Eu(3+) complex on the 5'-terminus and the other, labeled with a bidentate beta-diketone on the 3'-terminus. After hybridization of two DNA probes with target DNA, EDTA-Eu(3+) and beta-diketone come close to each other, and an EDTA-Eu(3+)-beta-diketonate ternary complex with a strong and long-lived fluorescence was formed; thus the target DNA was detected sensitively with a detection limit of 6 pM (0.6 fmol per assay) by time-resolved fluorescence measurement. In the absence of the target DNA, due to the poor stability of bidentate beta-diketonate-Eu(3+) complex in very diluted solution, only a small amount of ternary fluorescence complex was formed.     

The quenching of electrochemiluminescence upon oligonucleotide hybridization.

Many genomic assays rely on a distance-dependent interaction between luminescent labels, such as luminescence quenching or resonance energy transfer. We studied the interaction between electrochemically excited Ru(bpy)(3) (2+) and Cy5 in a hybridization assay on a chip. The 3' end of an oligonucleotide was labelled with Ru(bpy)(3) (2+) and the 5' end of a complementary strand with Cy5. Upon the hybridization, the electrochemiluminescence (ECL) of Ru(bpy)(3) (2+) was efficiently quenched by Cy5 with a sensitivity down to 30 nmol/L of the Cy5-labelled complementary strand. The quenching efficiency is calculated to be 78%. A similar phenomenon was observed in a comparative study using laser-excitation of Ru(bpy)(3) (2+). The hybridization with the non-labelled complementary or labelled non-complementary strand did not change the intensity of the ECL signal. Resonance energy transfer, electron transfer and static quenching mechanisms are discussed. Our results suggest that static quenching and/or electron transfer are the most likely quenching mechanisms.     

Solution-phase detection of polynucleotides using interacting fluorescent labels and competitive hybridization

DNA was assayed in a homogeneous format using DNA probes containing hybridization-sensitive labels. The DNA probes were prepared from complementary DNA strands in which one strand was covalently labeled on the 5'-terminus with fluorescein and the complementary strand was covalently labeled on the 3'-terminus with a quencher of fluorescein emission, either pyrenebutyrate or sulforhodamine 101. Probes prepared in this manner were able to detect unlabeled target DNA by competitive hybridization producing fluorescence signals which increased with increasing target DNA concentration. A single pair of complementary probes detected target DNA at a concentration of approximately 0.1 nM in 10 min or about 10 pM in 20-30 min. Detection of a 4 pM concentration of target DNA was demonstrated in 6 h using multiple probe pairs. The major limiting factors were background fluorescence and hybridization rates. Continuous monitoring of fluorescence during competitive hybridization allowed correction for variable sample backgrounds at probe concentrations down to 20 pM; however, the time required for complete hybridization increased to greater than 1 h at probe concentrations below 0.1 nM. A promising application for this technology is the rapid detection of amplified polynucleotides. Detection of 96,000 target DNA molecules in a 50-microliters sample was demonstrated following in vitro amplification using the polymerase chain reaction technique.     

HIV-1 protease: characterization of a catalytically competent enzyme-substrate intermediate.

The steady-state and pre-steady-state kinetic parameters for the interaction of E with the fluorogenic substrate 2-aminobenzoyl-Thr-Ile-Nle-Phe(p-NO(2))-Gln-Arg-NH(2) were determined in 1.25 M NaCl, 0.1 M MES-TRIS at pH 6.0 at 25 degrees C. At low concentrations of enzyme, the values of the K(m) and k(cat) calculated from steady-state data were 2.1 microM and 7.4 s(-1), respectively. At high concentrations of enzyme, the time-courses of fluorescence enhancement associated with catalysis were very dependent on the excitation wavelength used to monitor the reaction. Because the absorbance spectrum of the substrate overlapped the fluorescence emission spectrum of the enzyme, these abnormalities were attributed to fluorescence energy transfer between the enzyme and the substrate in an enzyme-substrate intermediate. The kinetic data collected with lambda(ex) = 280 nm and lambda(em) > 435 nm were analyzed according to the following mechanism in which EX was the species with enhanced fluorescence relative to substrate or products: [formula see text]. The values of the kinetic parameters with (1)H(2)O as the solvent were K = 13 microM, k(2) = 150 s(-1), k(-2) = 25 s(-1), and k(3) = 11 s(-1). The values of the kinetic parameters with (2)H(2)O as the solvent were K = 13 microM, k(2) = 210 s(-1), k(-2) = 12 s(-1), and k(3) = 4.4 s(-1). These values yielded solvent isotope effects of 2 on k(cat) and 0.9 on k(cat)/K(m). From analysis of the complete time-course of the fluorescence change (lambda(ex) = 280 nm and lambda(em) > 435 nm) during the course of substrate hydrolysis, the intermediate EX was determined to be 6.3-fold more fluorescent than the product, which, in turn, was 4.5-fold more fluorescent than ES or S. Rapid quench experiments with 2 N HCl as the quenching reagent confirmed that EX was a complex between enzyme and substrate. Consequently, the small burst in fluorescence observed when monitoring with lambda(ex) = 340 nm (0.3 product equiv per enzyme equivalent) was attributed to the fluorescence change upon transfer of substrate from an aqueous environment to a nonaqueous environment in the enzyme. These results were consistent with carbon-nitrogen bond cleavage being the major contributor to k(cat).     

Probing DNA sequences in solution with a monomer-excimer fluorescence color change.

The use of a simple fluorescent nucleoside analogue in detection of point mutations by hybridization in solution is described. Pyrene is placed at 3' and 5' ends of a pair of oligodeoxynucleotide probes via a phosphoramidite derivative of deoxyribose with this fluorophore attached at the 1' position, replacing a DNA base. Adjacent binding of dual probes containing this fluorophore to a complementary target sequence results in a pronounced spectral change from blue pyrene monomer emission (lambdamax= 381 398 nm) to green-white excimer emission (lambdamax= 490 nm). Optimization of the relative binding positions of the two probes shows that the greatest spectral change occurs when they bind with partial end overlap. In optimum orientation, the monomer emission band for the probes decreases intensity by as much as a factor of seven and the excimer band increases up to 40-fold on binding a complementary target. Application to the detection of a single-base point mutation in solution is described.     

Highly sensitive oligonucleotide-based fluorometric detection of mercury(II) in aqueous media

This paper describes a highly sensitive and selective Hg(2+) sensor using a label free Hg(2+) specific probe (5'-18T-3') and an intercalation dye SYBR Green I (SG). The Hg(2+) specific probe is composed of thymines (T) and readily forms T-Hg(2+)-T complexes in the presence of Hg(2+). This specific T-Hg(2+)-T formation affects the hybridization of the Hg(2+) specific probe and the intercalation of SG. Upon treatment of 1 nM 5'-18T-3' with different amount of Hg(2+) (0.1-10nM), which is followed by hybridization with 1 nM 5'-18T-3' and incubation with 1 microL of SG, the solution fluorescence gave a linear response (R=0.996) to the concentration of Hg(2+). The detection limit for Hg(2+) was 0.5 nM (0.1 ppb). The overall test only takes few minutes and very little interference is observed from non-specific metal ions. This approach may find potential applications in monitoring the Hg(2+) concentration in drinking water.     

Dimethyl-and monomethyloxyluciferins as analogs of the product of the bioluminescence reaction catalyzed by firefly luciferase

The absorption and fluorescence spectra of dimethyloxyluciferin (DMOL) and monomethyloxyluciferin (MMOL) were studied at pH 3.0-12.0. In the range of pH 3.0-8.0, the fluorescence spectrum of DMOL exhibits a maximum at lambda(em) = 639 nm. At higher pH values an additional emission maximum appears at lambda(em) = 500 nm (wavelength of excitation maximum lambda(ex) = 350 nm), which intensity increases with time. It is shown that this peak corresponds to the product of DMOL decomposition at pH > 8.0. The absorption spectra of MMOL were studied in the range of pH 6.0-9.0. At pH 8.0-9.0, the absorption spectrum of MMOL exhibits one peak at lambda(abs) = 440 nm. At pH 7.3-7.7, an additional band appears with maximum at lambda(abs) = 390 nm. At pH 6.0-7.0 two maxima are observed, at lambda(abs) = 375 and 440 nm. The fluorescence spectra of MMOL (pH 6.0-9.7, lambda(ex) = 440 or 375 nm) exhibit one maximum. It is shown that decomposition of DMOL and MMOL in aqueous solutions results in products of similar structure. DMOL and MMOL are rather stable at the pH optimum of luciferase. It is suggested that they can be used as fluorescent markers for investigation of the active site of the enzyme.     

Di(1,N6-ethenoadenosine)5', 5'-P1,P4-tetraphosphate, a fluorescent enzymatically active derivative of Ap4A

Di(1,N6-ethenoadenosine)5',5'-P1,P4-tetraphosphate, epsilon-(Ap4A), a fluorescent analog of Ap4A has been synthesized by reaction of 2-chloroacetaldehyde with Ap4A. At neutral pH this Ap4A analog presents characteristics maxima at 265 and 274 nm, shoulders at ca 260 and 310 nm and moderate fluorescence (lambda exc 307 nm, lambda em 410 nm). Enzymatic hydrolysis of the phosphate backbone produced a slight hyperchromic effect but a notorious increase of the fluorescence emission. Cytosolic extracts from adrenochromaffin tissue as well as cultured chromaffin cells were able to split epsilon(Ap4A) and catabolize the resulting epsilon-nucleotide moieties up to epsilon-Ado.     

Determination of alkaline phosphatase based on affinity adsorption solid-substrate room temperature phosphorimetry using rhodamine 6G-dibromoluciferin luminescent nanoparticle to label lectin and prediction of diseases

In the presence of heavy atom perturber LiAc, the silicon dioxide nanoparticle containing rhodamine 6G (R) and dibromoluciferin (D) (R-D-SiO(2)) can emit strong and stable solid-substrate room temperature phosphorescence signal of R (lambda(ex)/lambda(em)=481/648 nm) and D (lambda(ex)/lambda(em)=457/622 nm) on the surface of acetyl cellulose membrane (ACM). R-D-SiO(2) is used to label triticum vulgare lectin (WGA). Then two types of affinity adsorption reactions, R-D-SiO(2)-WGA- alkaline phosphatase (ALP) (direct method) and WGA-ALP-WGA-R-D-SiO(2) (sandwich method), are carried out on ACM. The conditions and the analytical characteristics for the determination of ALP using affinity adsorption solid-substrate room temperature phosphorimetry (AA-SS-RTP) were studied. For a 0.40-microl drop of sample, results show that the detection limits of the sandwich method are 0.16 ag spot(-1)(457/622 nm) and 0.17 ag spot(-1)(481/648 nm), and the detection limits of the direct method are 0.41 ag spot(-1) (457/622 nm) and 0.44 ag spot(-1) (481/648 nm). The contents of ALP in human serum correlated well with those obtained by enzyme-linked immunoassay. This study shows that AA-SS-RTP whether by the sandwich method or the direct method, can combine very well the characteristics of both high sensitivity of SS-RTP and specificity of the immunoreaction. Simultaneously, whether the phosphorescence excitation/emission wavelength of either R or D in R-D-SiO(2) is chosen to determine ALP, this can promote the agility and widen the adaptability of AA-SS-RTP.     

Sequence of methylated nucleotides at the 5''-terminus of adenovirus-specific RNA.

RNA labeled with [methyl-3H] methionine and [14C]uridine was isolated from the cytoplasm of adenovirus-infected cells and purified by poly(U)-Sepharose chromatography and hybridization to filters containing immobilized adeovirus DNA. Analysis by dimethyl sulfoxide-sucrose gradient sedimentation suggested that the major mRNA species were methylated. 7-Methylguanosine was identified at the 5'-terminus of the advenovirus-specific RNA and could be removed by periodate oxidation and beta-elimination. Structures of the type m7G(5')ppp(5')Nm containing the unusual nucleoside N6, O2'-dimethyladenosine, and smaller amounts of 2'-O-methyladenosine were isolated by DEAE-cellulose chromatography after P1 nuclease digestion of the RNA. Evidence for some 5'-terminal sequences, m7G(5')ppp(5')m6AmpNm, with additional 2'-O-methylribonucleosides was also obtained. A base-methylated nucleoside, N6-methyladenosine, is located within the RNA chain and is released as a mononucleotide by alkali hydrolysis.     

Single-nucleotide polymorphism analysis using fluorescence resonance energy transfer between DNA-labeling fluorophore,fluorescein isothiocyanate,and DNA intercalator,POPO-3, on bacterial magnetic particles

To develop an analytical system for single-nucleotide polymorphisms (SNPs), the fluorescence resonance energy transfer (FRET) technique was employed on a bacterial magnetic particle (BMP) surface. A combination of fluorescein isothiocyanate (FITC; excitation 490 nm/emission 520 nm) labeled at the 5' end of DNA and an intercalating compound (POPO-3, excitation 534 nm/emission 570 nm) was used to avoid the interference from light scattering caused by nanoparticles. After hybridization between target DNA immobilized onto BMPs and FITC-labeled probes, fluorescence from POPO-3, which was excited by the energy from the FITC, was detected. The major homozygous (ALDH2*1), heterozygous (ALDH2*1/*2), and minor homozygous (ALDH2*2) genotypes in the blood samples were discriminated by this method. The assay described herein allows for a simple and rapid SNP analysis using a fully automated system.     

Fluorescence and FT-IR spectroscopic studies of Suwannee River fulvic acid complexation with aluminum, terbium and calcium.

In this study fluorescence emission and IR spectroscopy have been used to investigate the interaction of the class A (oxygen seeking "hard acid") metal Al(3+), with Suwannee River fulvic acid. Addition of Al(3+) ion results in a significant enhancement in fulvic acid fluorescence emission (at lambda(em)=424 nm) and significant red shift of the excitation wavelength (from lambda(ex)=324 nm to lambda(ex)=344 nm) at low pH values (pH approximately 4.0-5.0). At pH 4.0 (0.1 M ionic strength), where the predominant aluminum ion species is the "free" (aquo) ion, the fulvic acid fluorescence reaches 142% of the value in the absence of added metal ion. Analysis of the pH 4.0 and pH 5.0 fluorescence enhancement data with the nonlinear (single site) model of Ryan and Weber indicated binding constants in the range of 4.67.10(4)-2.87.10(6) M(-1) and concentrations of ligand sites in the range of 18.6.10(-6)-24.0.10(-6) M, both consistent with previous studies performed on both aquatic and soil fulvic acids. Companion fluorescence experiments performed on two other class A metal ions, Ca(2+) and Tb(3+), indicated no significant enhancement or quenching with Ca(2+) and only slight quenching with Tb(3+). Comparison of FT-IR spectra collected on fulvic acid alone and fulvic acid in the presence of the three class A metals (Al(3+), Ca(2+) and Tb(3+)) provides strong evidence for the involvement of carboxyl carbonyl functions in the binding of all three metal ions, which is not unexpected. The spectra also reveal, however, a very pronounced difference in the 4000-2000 cm(-1) IR spectral region between the Al(3+) spectrum and the Ca(2+) and Tb(3+) spectra. The -OH stretch spectral region in the Al(3+) spectrum has a major component shifted to higher energy (compared to fulvic acid alone or to fulvic acid in the presence of Ca(2+) or Tb(3+)). Even more striking is the emergence of a pronounced IR band at 2407 cm(-1), which is present only in the Al(3+) spectrum. The results of fluorescence and IR experiments with the model compounds salicylic acid and phthalic acid further confirm that both salicylic acid-like sites and phthalic acid-like sites are likely complexation sites for Al(3+) in fulvic acid and are major contributors to the observed spectroscopic changes associated with Al(3+) ion complexation. From a comparison of both the fluorescence and IR spectral results for all three class A metals, differing most strongly in the value of their ionic index, it seems clear that major sources of the deviation in spectral properties between Al(3+) and Ca(2+)/Tb(3+) is the unusually high value of its charge density and relatively low propensity for involvement in covalent bonding interactions (very high ionic index and relatively low covalent index in the Nieboer and Richardson classification of environmental metals), as well as affinity for certain functional groups.     

Tunable blinking kinetics of cy5 for precise DNA quantification and single-nucleotide difference detection

Fluorescence correlation spectroscopy (FCS) can resolve the intrinsic fast-blinking kinetics (FBKs) of fluorescent molecules that occur on the order of microseconds. These FBKs can be heavily influenced by the microenvironments in which the fluorescent molecules are contained. In this work, FCS is used to monitor the dynamics of fluorescence emission from Cy5 labeled on DNA probes. We found that the FBKs of Cy5 can be tuned by having more or less unpaired guanines (upG) and thymines (upT) around the Cy5 dye. The observed FBKs of Cy5 are found to predominantly originate from the isomerization and back-isomerization processes of Cy5, and Cy5-nucleobase interactions are shown to slow down these processes. These findings lead to a more precise quantification of DNA hybridization using FCS analysis, in which the FBKs play a major role rather than the diffusion kinetics. We further show that the alterations of the FBKs of Cy5 on probe hybridization can be used to differentiate DNA targets with single-nucleotide differences. This discrimination relies on the design of a probe-target-probe DNA three-way-junction, whose basepairing configuration can be altered as a consequence of a single-nucleotide substitution on the target. Reconfiguration of the three-way-junction alters the Cy5-upG or Cy5-upT interactions, therefore resulting in a measurable change in Cy5 FBKs. Detection of single-nucleotide variations within a sequence selected from the Kras gene is carried out to validate the concept of this new method.     

Perturbing the DNA sequence selectivity of metallointercalator-peptide conjugates by single amino acid modification.

Metallointercalator-peptide conjugates that provide small molecular mimics to explore peptide-nucleic acid recognition have been prepared. Specifically, a family of peptide conjugates of [Rh(phi)(2)(phen')](3+) [where phi = 9,10-phenanthrenequinone diimine and phen' = 5-(amidoglutaryl)-1,10-phenanthroline] has been synthesized and their DNA-binding characteristics examined. Single amino acid modifications were made from the parent metallointercalator-peptide conjugate [Rh(phi)(2)(phen')](3+)-AANVAIAAWERAA-CONH(2), which targets 5'-CCA-3' site-specifically. Moving the glutamate at position 10 in the sequence of the appended peptide to position 6 {[Rh(phi)(2)(phen')](3+)-AANVAEAAWARAA-CONH(2)} changed the sequence preference of the metallointercalator-peptide conjugate to 5'-ACA-3'. Subsequent mutation of the glutamate at position 6 to arginine {[Rh(phi)(2)(phen')](3+)-AANVARAAWARAA-CONH(2)} caused more complex changes in DNA recognition. Thermodynamic dissociation constants were determined for these metallointercalator-peptide conjugates by photoactivated DNA cleavage assays with the rhodium intercalators. At 55 degrees C in the presence of 5 mM MnCl(2), [Rh(phi)(2)(phen')](3+)-AANVAIAAWERAA-CONH(2) binds to a 5'-CCA-3' site with K(d) = 5.7 x 10(-)(8) M, whereas [Rh(phi)(2)(phen')](3+)-AANVAEAAWARAA-CONH(2) binds to its target 5'-ACA-3' site with K(d) = 9.9 x 10(-8) M. The dissociation constant for [Rh(phi)(2)(phen')](3+) with random-sequence DNA is 7.0 x 10(-7) M. Structural models have been developed and refined to account for the observed sequence specificities. As with much larger DNA-binding proteins, with these metal-peptide conjugate mimics, single amino acid changes can lead to single or multiple base changes in the DNA site targeted.     

Aminoquinolines as fluorescent labels for hydrophilic interaction liquid chromatography of oligosaccharides

Abstract In this study, we investigated the potential of four different aminoquinoline (AQ) compounds as fluorescent labels for glycan analysis using hydrophilic interaction liquid chromatography (HILIC) and fluorescence detection (FLD). We confirmed the optimal excitation and emission wavelengths of 3-AQ and 6-AQ conjugated to glycan standards using three-dimensional fluorescent spectral scanning. The optimal excitation and emission wavelengths for 6-AQ were confirmed at λex=355 nm and λem=440 nm. We concluded that the optimal wavelengths for 3-AQ were λex=355 nm and λem=420 nm, which differed considerably from the wavelengths applied in previous reports. HILIC-FLD chromatograms using experimentally determined wavelengths were similar to 2-aminobenzamide controls, but the peak capacity and resolution differed significantly when published 3-AQ λex/em values were applied. Furthermore, we found that 5-AQ and 8-AQ labeled maltohexaose did not display any fluorescent pro\xadperties when used as a carbohydrate tag for HPLC analysis. Finally, we applied experimentally determined wavelengths to 3-AQ labeled N-glycans released from human IgG to illustrate changes in retention time as well as to demonstrate that AQ labeling is applicable to complex sample analysis via exoglycosidase sequencing.     

Fluorescence properties of free and protein bound fluorescein dyes. I. Macrospectrofluorometric measurements.

Excitation and emission properties of fluorescein derivatives were studied macrofluorometrically. Measurements were performed with solutions of various concentrations (0.07-100 microgram/ml) of free sodium fluorescein prepared from fluorescein diacetate (FDA), fluorescein isothiocyanate (FITC) and FITC bound to rabbit gamma-globulin. Both excitation and emission spectra as well as fluorescence intensities at constant excitation/emission wavelengths (496/515 nm) were recorded. The findings indicate that (1) FDA gives about twice the fluorescence intensity compared to equal concentrations of FITC. (2) The fluorescence properties of FITC upon excitation with blue light (lambda = 496 nm) are only slightly altered by the conjugation to rabbit gamma-globulin. (3) Considerable quenching due to conjugation could, however, be shown to occur upon UV excitation (lambda = 340 nm). (4) Fluorescence emission excited by visible blue light (496 nm) increases linearly to dye concentration in a range of 0.07-2.5 microgram/ml. Beginning at 5 microgram/ml (10-(5) M/1) all three compounds show a sharp decrease of fluorescence intensity with further increasing concentration. Practical aspects of these data for the immunofluorescence method are discussed.     

Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates.

Alexa 350, Alexa 430, Alexa 488, Alexa 532, Alexa 546, Alexa 568, and Alexa 594 dyes are a new series of fluorescent dyes with emission/excitation spectra similar to those of AMCA, Lucifer Yellow, fluorescein, rhodamine 6G, tetramethylrhodamine or Cy3, lissamine rhodamine B, and Texas Red, respectively (the numbers in the Alexa names indicate the approximate excitation wavelength maximum in nm). All Alexa dyes and their conjugates are more fluorescent and more photostable than their commonly used spectral analogues listed above. In addition, Alexa dyes are insensitive to pH in the 4-10 range. We evaluated Alexa dyes compared with conventional dyes in applications using various conjugates, including those of goat anti-mouse IgG (GAM), streptavidin, wheat germ agglutinin (WGA), and concanavalin A (ConA). Conjugates of Alexa 546 are at least twofold more fluorescent than Cy3 conjugates. Proteins labeled with the Alexa 568 or Alexa 594 dyes are several-fold brighter than the same proteins labeled with lissamine rhodamine B or Texas Red dyes, respectively. Alexa dye derivatives of phalloidin stain F-actin with high specificity. Hydrazide forms of the Alexa dyes are very bright, formaldehyde-fixable polar tracers. Conjugates of the Alexa 430 (ex 430 nm/em 520 nm) and Alexa 532 (ex 530 nm/em 548 nm) fluorochromes are spectrally unique fluorescent probes, with relatively high quantum yields in their excitation and emission wavelength ranges.     

UV irradiation of nucleic acids: formation, purification and solution conformational analysis of the '6-4 lesion' of dTpdT

Irradiation of dTpdT with 300 kJ/m2 of 254 nm produces numerous photo-products, one of which labeled dT6pd4T[1] was purified by HPLC. dT6pd4T has a UV spectrum (H20, pH 7) with lambda max = 326 nm and lambda min = 265 nm, and a P-31 NMR resonance at -3.46 ppm (normal dTpdT occurs at -4.01 ppm; TMP, 30 degrees C). 2-D COSY NMR spectra facilitated proton resonance assignments and 2-D NOESY spectra aided analysis of spatial orientation. Carbon-13 and proton-coupled P-31 NMR spectra of dT6pd4T were also obtained. These analyses indicate: C5=C6 of dT6p- is saturated and the -pd4T base is more aromatic; the dT6p- base possesses a configuration of 5R, 6S; dT6p- and -pd4T have anti-type glycosidic conformations; furanose conformation of dT6p- is mainly C3'-endo and that of -pd4T exists in a C3'-endo in equilibrium C3'-exo; exocyclic bonds gamma (C5'-C4'), beta (05'-C5') and epsilon (C3'-03') are non-classical rotamers; dihedral angle about epsilon (C3'-03') is smaller relative to dTpdT.     

Stability, specificity and fluorescence brightness of multiply-labeled fluorescent DNA probes.

In this work, we studied the fluorescence and hybridization of multiply-labeled DNA probes which have the hydrophilic fluorophore 1-(straightepsilon-carboxypentynyl)-1'-ethyl- 3,3,3', 3'-tetramethylindocarbocyanine-5,5'-disulfonate (Cy3) attached via either a short or long linker at the C-5 position of deoxyuridine. We describe the effects of labeling density, fluorophore charge and linker length upon five properties of the probe: fluorescence intensity, the change in fluorescence upon duplex formation, the quantum yield of fluorescence (Phif), probe-target stability and specificity. For the hydrophilic dye Cy3, we have demonstrated that the fluorescence intensity andPhifare maximized when labeling every 6th base using the long linker. With a less hydrophilic dye, a labeling density this high could not be achieved without serious quenching of the fluorescence. The target specificity of multiply-labeled DNA probes was just as high as compared to the unmodified control probe, however, a less stable probe-target duplex is formed that exhibits a lower melting temperature. A mechanism that accounts for this destabilization is proposed which is consistent with our data. It involves dye-dye and dye-nucleotide interactions which appear to stabilize a single-stranded conformation of the probe.