Coupling a natural receptor protein with an artificial receptor to afford a semisynthetic fluorescent biosensor

An artificial receptor and a signal transducer have been engineered on a lectin (saccharide-binding protein) surface by a post-photoaffinity labeling modification method. Saccharide binding can be directly and selectively read out by the fluorescence changes of the fluorophore via photoinduced electron transfer (PET) mode. Fluorescence titration with various saccharides reveals that molecular recognition by the artificial receptor is successfully coupled to the native binding site of the lectin, producing a novel fluorescent saccharide biosensor showing modulated specificity and enhanced affinity. Designed cooperativity between artificial and native molecular recognition modules was quantitatively demonstrated by the comparison of the binding affinities, and it represents a new strategy in molecular recognition. By using appropriate artificial receptors and various native lectins, this approach may provide many new semisynthetic biosensors for saccharide derivatives such as glycolipids and glycopeptides/proteins. An extended library of lectin-based biosensors is envisioned to be useful for glycome research, a newly emerging field of the post-genomic era.     

Double-modification of lectin using two distinct chemistries for fluorescent ratiometric sensing and imaging saccharides in test tube or in cell

The site-selective incorporation of two different fluorophores into a naturally occurring protein (lectin, a sugar-binding protein) has been successfully carried out using two distinct orthogonal chemical methods. By post-photoaffinity labeling modification, Con A, a glucose- and mannose-selective lectin, was modified with fluorescein in the proximity of the sugar binding site (Tyr100 site), and the controlled acylation reaction provided the site-selective attachment of coumarin at Lys114. In this doubly modified Con A, the fluorescein emission changed upon the binding to the corresponding sugars, such as the glucose or mannose derivatives, whereas the coumarin emission was constant. Thus, the doubly modified Con A fluorescently sensed the glucose- and mannose-rich saccharides in a ratiometric manner while retaining the natural binding selectivity and affinity, regardless of the double modification. On the benefit of the ratiometric fluorescent analysis using two distinct probes, the sugar trimming process of a glycoprotein can be precisely monitored by the engineered Con A. Furthermore, the doubly modified Con A can be used not only for the convenient fluorescent imaging of saccharides localized on a cell surface, such as the MCF-7, a breast cancer cell having rich high-mannose branch, but also for the ratiometric fluorescent sensing of the glucose concentration inside HepG2 cells. These results demonstrated that the semisynthetic lectin modified doubly by two distinct chemistries is superior to the singly modified one in function, and thus, it may be potentially useful in cell, as well as in test tube.     

Differences in the redistribution of concanavalin A and wheat germ agglutinin binding sites on mouse neuroblastoma cells.

Concanavalin A (Con A), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA) bound with either 125I, fluorescent dyes, or fluorescent polymeric microspheres were used to quantitate and visualize the distribution of lectin binding sites on mouse neuroblastoma cells. As viewed by fluorescent light and scanning electron microscopy, over 10(7) binding sites for Con A, WGA, and RCA appeared to be distributed randomly over the surface of differentiated and undifferentiated cells. An energy-dependent redistribution of labeled sites into a central spot occurred when the cells were labeled with a saturating dose of fluorescent lectin and maintained at 37 degrees C for 60 min. Reversible labeling using appropriate saccharide inhibitors indicated that the labeled sites had undergone endocytosis by the cell. A difference in the mode of redistribution of WGA or RCA and Con A binding sites was observed in double labeling experiments. When less than 10% of the WGA or RCA lectin binding sites were labeled, only these labeled sites appeared to be removed from the cell surface. In contrast, when less than 10% of the Con A sites were labeled, both labeled and unlabeled Con A binding sites were removed from the cell surface. Cytochalasin B uncoupled the coordinate redistribution of labeled and unlabeled Con A sites, suggesting the involvement of microfilaments. Finally, double labeling experiments employing fluorescein-tagged Con A and rhodamine-tagged WGA indicate that most Con A and WGA binding sites reside on different membrane components and redistribute independenty of each other.     

Fluorophore appended saccharide cyclophane: self-association, fluorescent properties, heterodimers with cyclodextrins, and cross-linking behavior with peanut agglutinin of dansyl-modified saccharide cyclophane

A saccharide cyclophane bearing an environment-sensitive fluorophore (1) was prepared by introducing not only three branches with a terminal galactose residue but also one with a dansyl moiety into a tetraaza[6.1.6.1]paracyclophane skeleton. Self-association behavior of the dansyl-appended saccharide cyclophane was characterized in aqueous media by fluorescence spectroscopy and dynamic light scattering measurements. At least in the concentrations below 1.0 x 10(-5) M, saccharide cyclophane 1 existed in a monomeric state, whereas it tended to form self-aggregated complexes in the higher concentration. Solvent polarity dependency on the emission spectra of 1 was examined by fluorescence spectroscopy. With increasing dioxane contents in dioxane/water solvents, the fluorescence intensity originating from the dansyl moiety of 1 increased along with a concomitant blue shift of the fluorescence maximum (lambda(em)). In the monomeric state of 1 in water, the dansyl moiety of 1 was not fully included into its cyclophane cavity but partially exposed to the bulk aqueous phase. In the higher concentration ranges in an aggregate state, however, the dansyl group of 1 was located in the apolar cyclophane cavity whose microenvironment was equivalent to the polarity of 1-butanol evaluated on the basis of a correlation between lambda(em) and solvent polarity. This indicates an intermolecular inclusion of the dansyl moiety within the cyclophane. When cyclodextrin (CD) was mixed with 1, the dansyl group of 1 was bound to an internal cavity of CD such as gamma-CD, beta-CD, 6-O-alpha-glucosyl-beta-CD, and 6-O-alpha-maltosyl-beta-CD with binding constants of 7.5 x 10(2), 7.8 x 10(2), 7.7 x 10(2), and 6.0 x 10(2) M(-1), respectively. Such a supramolecular assembling of dansyl-modified cyclophane 1 and CDs caused changes of the fluorescence spectra as well as appearance of induced CD bands in aqueous media. Furthermore, saccharide cyclophane 1 was selectively bound to peanut agglutinin (PNA), galactoside-binding lectin, which was readily monitored by a visible turbidity of the solution due to a cross-linking agglutination of these components, as well as by fluorescence spectroscopy.     

Amide-based molecular knots as platforms for fluorescent switches

A series of amide-based molecular knots equipped selectively with fluorescent dansyl and/or pyrenesulfonyl moieties were synthesized from the readily available tris(allyloxy)knotane. UV/Vis absorption spectra, emission spectra, and the emission lifetimes of the fluorescent knotanes were investigated in chloroform at 298 K. The absorption spectra of the knotanes correspond to those of mixtures of their UV-active constituents. The fluorescence quantum yields and lifetimes of the dansyl and pyrenesulfonyl moieties are partly quenched by the knotane platform. In the KN(Da)(2)(Py) species, the fluorescent excited state of the dansyl units (lambda(max)=510 nm) lies at lower energy than the fluorescent excited state of the pyrenesulfonyl unit (lambda(max)=385 nm), the emission of which is accordingly quenched with sensitization of the dansyl fluorescence. In the KN(Ao)(2)(Da), KN(Ao)(Da)(2), and KN(Da)(3) species, the addition of acids causes the protonation of their dansyl units with a consequent decrease in the intensity of the dansyl band at 510 nm and appearance of the emission band of the protonated dansyl unit (lambda(max)=340 nm). Each dansyl unit of KN(Ao)(Da)(2) and KN(Da)(3) undergoes the independent protonation. In these incompletely protonated knotanes the fluorescence of the protonated dansyl units is partly quenched by nonprotonated ones. These processes can be quantitatively reversed upon addition of a base. In KN(Da)(2)(Py), an increase of the fluorescence of its pyrenesulfonyl group is observed when the dansyl groups are protonated. The results obtained show that the readily available and easily functionalizable amide-knotanes can be used as an interesting scaffold to obtain fluorescent switches.     

Fluorescence Turn‐On Sensing of Lectins with Mannose‐Substituted Tetraphenylethenes Based on Aggregation‐Induced Emission

The synthesis of mannose‐substituted tetraphenylethenes (TPEs) and their aggregation‐induced emission (AIE) behavior, induced by interactions with concanavalin A (Con A), are reported. A mixture of the mannose‐TPE conjugates and Con A in a buffer solution displays an intense blue emission on agglutination within a few seconds, which serves as a “turn‐on” fluorescent sensor for lectins. The sensing is also selective: the conjugates act as a sensor for Con A, but do not sense a galactose‐binding lectin, PNA. Con A‐recognition is not affected even in the presence of other proteins in a mixture. The conjugates also exhibit high sensitivity to detect Con A. An increased sensitivity of the conjugates results if mannopyranoside substituents are linked to the TPE‐core unit with a flexible chain and/or when the number of mannose residues increases.     

Mechanisms for fluorescence depolarization in dendrimers

We have investigated the fluorescence properties of dendrimers (Gn is the dendrimer generation number) containing four different luminophores, namely terphenyl (T), dansyl (D), stilbenyl (S), and eosin (E). In the case of T, the dendrimers contain a single p-terphenyl fluorescent unit as a core with appended sulfonimide branches of different size and n-octyl chains. In the cases of D and S, multiple fluorescent units are appended in the periphery of poly(propylene amine) dendritic structures. In the case of E, the investigated luminophore is noncovalently linked to the dendritic scaffold, but is encapsulated in cavities of a low luminescent dendrimer. Depending on the photophysical properties of the fluorescent units and the structures of the dendrimers, different mechanisms of fluorescence depolarization have been observed: (i) global rotation for GnT dendrimers; (ii) global rotation and local motions of the dansyl units at the periphery of GnD dendrimers; (iii) energy migration among stylbenyl units in G2S; and (iv) restricted motion when E is encapsulated inside a dendrimer, coupled to energy migration if the dendrimer hosts more than one eosin molecule.     

A 'turn-on' FRET peptide sensor based on the mercury binding protein MerP

A new fluorescent peptidyl chemosensor based on the mercury binding MerP protein with fluorescence resonance energy transfer (FRET) capabilities has been synthesized via Fmoc solid-phase peptide synthesis. The metal chelating unit, which is flanked by the fluorophores tryptophan (donor) and dansyl (acceptor), contains amino acids from MerP's metal binding loop (sequence: dansyl-Gly-Gly-Thr-Leu-Ala-Val-Pro-Gly-Met-Thr-Cys-Ala-Ala-Cys-Pro-Ile-Thr-Val-Lys-Lys-Gly-Gly-Trp-CONH(2)). A FRET enhancement or 'turn-on' response was observed for Hg(2+) as well as for Zn(2+), Cd(2+) and Ag(+) in a pure aqueous solution at pH 7.0. The emission intensity of the acceptor was used to monitor the concentration of these metals ions with detection limits of 280, 6, 103 and 496 microg L(-1), respectively. No response was observed for the other transition, alkali and alkaline earth metals tested. The fluorescent enhancement observed is unique for Hg(2+) since this metal generally quenches fluorescence. The acceptor fluorescence increase resulting from metal binding-induced FRET suggests a sensor that is inherently more sensitive than one based on quenching by the binding event.     

Bi-fluorescence-labeled maltoheptaoside: convenient substrate for continual assay of alpha-amylase

A new maltoheptaose derivative was prepared as a useful substrate for continual assay of alpha-amylase. The maltoheptaoside has thionaphtyl group as a fluorescent energy donor at the reducing end and dansyl group as an acceptor group at the non-reducing end. Excitation of the thionaphthyl group at 290 nm results in emission at 523 nm from the dansyl group, while the emission from the thionaphthyl group is quenched by the dansyl group. This fluorescence energy transfer is reduced by the hydrolytic action with alpha-amylase and a significant decrease in the dansyl emission concomitant with an increase in the thionaphthyl emission was observed. Usefulness of this substrate was demonstrated for sensitive and continuous assay of alpha-amylase from Aspergillus oryzae.     

Fluorescent peptide sensor for the selective detection of Cu

A new fluorescent peptidyl chemosensor for Cu²⁺ ions with fluorescence resonance energy transfer (FRET) capabilities has been synthesized via Fmoc solid-phase peptide synthesis. The metal chelating unit, which is flanked by the fluorophores tryptophan (donor) and dansyl chloride (acceptor), consists of the amino acids glycine and aspartic acid (Gly-Gly-Asp-Gly-Gly-Asp-Gly-Gly-Asp-Gly-Gly-Asp-Gly-Gly). Coordination of the Cu²⁺ ions to the metal chelating unit results in fluorescent quenching of both the donor and acceptor fluorophores. Although it was determined that Cu²⁺ binding causes no change in FRET efficiency, emission and Cu²⁺-induced quenching of the acceptor dye can be used to monitor the concentration of the copper ions, with a detection limit of 32 μg L⁻¹. The sensor also demonstrated sensitivity, reversibility and selectivity towards Cu²⁺ in a transition metal matrix at pH 7.0.     

Enantioselective fluorescence sensing of amino acids by modified cyclodextrins: role of the cavity and sensing mechanism

Two selectors based on modified cyclodextrins containing a metal binding site and a dansyl fluorophore-6-deoxy-6-N-(N(alpha)-[(5-dimethylamino-1-naphthalenesulfonyl)aminoethyl]phenylalanylamino-beta-cyclodextrin-containing D-Phe (3) and L-Phe (4) moieties were synthesized. The conformations of the two selectors were studied by circular dichroism, two-dimensional NMR spectroscopy and time-resolved fluorescence spectroscopy. Cyclodextrin 4 was found to have a predominant conformation in which the dansyl group is self-included in the cyclodextrin cavity, while 3 showed a larger proportion of the conformation with the dansyl group outside the cavity. As a consequence, the two cyclodextrins were found to bind copper(II) with different affinities, as revealed by fluorescence quenching in competitive binding measurements. Addition of D- or L-amino acids induced increases in fluorescence intensity, which were dependent on the amino acid used and in some cases on its absolute configuration. The cyclodextrin 4 was found to be more enantioselective than 3, suggesting that the self-inclusion in the cyclodextrin cavity strongly increases the chiral discrimination ability of the copper(II) complex. Accordingly, a linear fluorescent ligand N(alpha)-[(5-dimethylamino-1-naphthalenesulfonyl)aminoethyl]-N(1)-propyl-phenylalaninamide, which has the same binding site and absolute configuration as 4, showed very low chiral discrimination ability. The enantioselectivity in fluorescence response was found to be due to the formation of diastereomeric ternary complexes, which were detected by ESI-MS and by circular dichroism. Time-resolved fluorescence studies showed that the fluorescence of the dansyl group was completely quenched in the ternary complexes formed, and that the residual fluorescence was due to uncomplexed ligand.     

Probing carbohydrate-lectin recognition in heterogeneous environments with monodisperse cyclodextrin-based glycoclusters

A series of β-cyclodextrin (βCD)-scaffolded glycoclusters exposing heterogeneous yet perfectly controlled displays of α-mannosyl (α-Man) and β-lactosyl (β-Lact) antennas were synthesized to probe the mutual influence of varying densities of the saccharide motifs in the binding properties toward different plant lectins. Enzyme-linked lectin assay (ELLA) data indicated that the presence of β-Lact residues reinforced binding of α-Man to the mannose-specific lectin concanavalin A (Con A) even though homogeneous β-Lact clusters are not recognized at all by this lectin, supporting the existence of synergic recognition mechanisms (heterocluster effect). Conversely, the presence of α-Man motifs in the heteroglycoclusters also resulted in a binding-enhancing effect of β-Lact toward peanut agglutinin (PNA), a lectin strongly binding multivalent lactosides but having no detectable affinity for α-mannopyranosides, for certain architectural arrangements. Two-site, sandwich-type ELLA data corroborated the higher lectin clustering efficiency of heterogeneous glycoclusters compared with homogeneous displays of the putative sugar ligand with identical valency. A turbidity assay was also consistent with the previous observations. Most revealingly, the lectin cross-linking ability of heterogeneous glycoclusters was sensitive to the presence of high concentrations of the non-ligand sugar, strongly suggesting that "mismatching" saccharide motifs may modulate carbohydrate-lectin specific recognition in a lectin-dependent manner when present in highly dense displays together with the "matching" ligand, a situation frequently encountered in biological systems.     

Fluorescent aromatic polyamides with urea binding sites and fluorene or dansyl signaling units

This work describes six novel fluorescent aromatic polyamides with bulky dansyl or fluorene pendant groups or with the fluorene moiety in the main chain. The fluorescent signaling dansyl or fluorene moieties are chemically connected to, or within, the main polymer chains through a urea group, a well-known binding site in supramolecular chemistry. These polyamides are amorphous and soluble in polar aprotic solvents and demonstrate a film-forming capability. They also show yellowish-green or blue fluorescence in solution and in the solid state depending on the signaling unit, with the former corresponding to the dansyl and the latter to the fluorene residue. The water uptake and the thermal behavior have also been evaluated and related to the chemical structure. The polar amide, urea, and sulfonamide groups give rise to a high water sorption of up to 3.8 water molecules per repeating unit. The thermal behavior has been investigated by means of DSC and TGA. The glass transition temperatures of the polymers are high (up to 331 °C) and the decomposition temperatures (around 300 °C) are due to the moderate thermal stability of the urea group.     

Clustering of Escherichia coli type-1 fimbrial adhesins by using multimeric heptyl α-D-mannoside probes with a carbohydrate core

Heptyl α‐D ‐mannoside (HM) is a strong inhibitor of the FimH lectin that mediates the initial adhesion of the uropathogenic Escherichia coli (E. coli) to the bladder cells. We designed a set of multivalent HM ligands based on carbohydrate cores with structural valencies that range from 1 to 7. The chemical strategy used to construct the regular hydrophilic structures consisted of the repetition of a critical glucoside fragment. A primary amino group was grafted at the sugar reducing end to couple the multimers to a fluorescent label. A one‐pot synthetic approach was developed to tether the ligands and the fluorescein isothiocyanate (FITC) probe to the scaffold simultaneously. Isothermal calorimetry with the monomeric FimH lectin revealed nanomolar affinities and saturation of all structurally available binding sites on the multivalent HM ligands. Direct titrations domain showed almost strict correlation of enthalpy–entropy compensation with increasing valency of the ligand, whereas reverse titration calorimetry demonstrated negative cooperativity between the first and the second binding site of the divalent heptyl mannoside. A multivalency effect was nevertheless observed by inhibiting the haemagglutination of type‐1 piliated UTI89 E. coli, with a titer as low as 60 nM for the heptavalent HM ligand. An FITC‐labeled HM trimer showed capture and cross‐linking of living bacteria in solution, a phenomenon not previously described with low‐valency ligands.     

Multi-mannosides based on a carbohydrate scaffold: synthesis, force field development, molecular dynamics studies, and binding affinities for lectin Con A

A short and efficient strategy for the synthesis of multi-valent mannosides based on a selectively functionalized carbohydrate scaffold was reported involving (i) direct regioselective azidation of unprotected commercial saccharides, (ii) acetylation, (iii) grafting of the mannosyl ligands by click chemistry, and (iv) deacetylation. New glycoclusters with a valency ranging from 1 to 4 and different spatial arrangements of the epitopes were obtained. Binding affinities of the new glycoclusters toward concanavalin A (Con A) lectin were investigated by an enzyme-linked lectin essay (ELLA). The synthetic multi-valent compounds exhibited a remarkable cluster effect with a relative potency per mannoside residue ranging from 8.1 to 9.1 depending on the structures. ELLA experiments were in agreement with the establishment of favorable interactions between triazole ring and Con A, increasing the binding affinity. A new force field topology database was developed in agreement with the GLYCAM 2004 force field. Molecular dynamics performed on representative glyco-conjugates revealed interesting structural features such as rigidity of the scaffold for a well-defined presentation of the ligands and highly flexible mannose counterparts. The new glycoconjugates reported may be promising tools as probes or effectors of biological processes involving lectins.     

A fluorescent lectin array using supramolecular hydrogel for simple detection and pattern profiling for various glycoconjugates

Because sugar and its derivatives play important roles in various biological phenomena, the rapid and high-throughput analysis of various glycoconjugates is keenly desirable. We describe herein the construction of a novel fluorescent lectin array for saccharide detection using a supramolecular hydrogel matrix. In this array, the fluorescent lectins were noncovalently fixed under semi-wet conditions to suppress the protein denaturation. It is demonstrated by fluorescence titration and fluorescence lifetime experiments that the immobilized lectins act as a molecular recognition scaffold in the hydrogel matrix, similar to that in aqueous solution. That is, a bimolecular fluorescence quenching and recovery (BFQR) method can successfully operate under both conditions. This enables one to fluorescently read-out a series of saccharides on the basis of the recognition selectivity and affinity of the immobilized lectins without tedious washing processes and without labeling the target saccharides. Simple and high-throughput sensing and profiling were carried out using the present lectin array for diverse glycoconjugates, which not only included a simple glucose, but also oligosaccharides, and glycoproteins, and, furthermore, the pattern recognition and profiling of several types of cell lysates were also accomplished.     

Cyclam‐Cored Dendrimers Appended with Four Dendrons of Two Different Types: Intradendrimer Energy Transfer

We have synthesized two cyclam‐cored dendrimers appended with dendrons of two different types by proper protection/deprotection of the cyclam unit. The resulting dendrimers contain six naphthyl and two dansyl units ( N6 D2 ) or two dansyl and six naphthyl units ( N2 D6 ) at the periphery. Their photophysical properties have been compared to those of a dendrimer containing 8 dansyl units ( D8 ) and a previously investigated dendrimer containing 8 naphthyl units ( N8 ). The absorption spectra are those expected on the basis of the number of chromophores, demonstrating that no ground state interaction takes place. The emission spectra of N2 D6 and N6 D2 show naphthalene localized and naphthalene excimer emission similar to those observed in the case of N8 , together with a much stronger dansyl emission with maximum at 525 nm. Addition of CF₃SO₃H to dendrimer solutions in CH₃CN/CH₂Cl₂ 1:1 (v/v) leads to protonation of the aliphatic amine units of the cyclam core at first and then of the aromatic amine of each dansyl chromophores. Cyclam can be diprotonated and this affects dansyl absorption and, most significantly, emission bands by a charge perturbation effect. Each dansyl unit is independently protonated in both dendrimers. The most interesting photophysical feature of these heterofunctionalized cyclam‐cored dendrimers is the occurrence of an intradendrimer photoinduced energy transfer from naphthyl to dansyl chromophores of two different dendrons (interdendron mechanism). The efficiency of this process is 50 % for N6 D2 and it can be increased up to 75 % upon protonation of the cyclam core and formation of N6 D2 (2H⁺). This arises from the fact that protonation of the amine units of the cyclam prevents formation of exciplexes upon naphthyl excitation, thus shutting down one of the deactivation processes of the fluorescent naphthyl excited state.     

The effect of the N-B transition of bovine and human serum albumins on the binding sites of fluorescent probes

The N(neutral)-B(alkaline) transition of bovine serum albumin (BSA) and human serum albumin (HSA) was investigated with three anionic and one cationic dansyl (dimethylamino-naphthalenesulfonyl, DNS) probes. The N-B transition of the HSA-probe complex was characterized by a large enhancement of the fluorescence intensity of the dansyl group, accompanied by a blue-shift of the maximum wavelength in the emission spectra, and large changes in the thermodynamic parameters of the binding process. Contrary to this, such changes were not observed for the BSA-probe complex. It is suggested that the conformational adaptability of HSA to the probes at the binding site increases in the B conformation, although the nature of the site on BSA is hardly affected by the transition.     

Determination of Fucose Concentration in a Lectin-Based Displacement Microfluidic Assay

We compare three different methods to quantify the monosaccharide fucose in solutions using the displacement of a large glycoprotein, lactoferrin. Two microfluidic analysis methods, namely fluorescence detection of (labeled) lactoferrin as it is displaced by unlabeled fucose and the displacement of (unlabeled) lactoferrin in SPR, provide fast responses and continuous data during the experiment, theoretically providing significant information regarding the interaction kinetics between the saccharide groups and binding sites. For comparison, we also performed a static displacement ELISA. The stationary binding site in all cases was immobilized S2-AAL, a monovalent polypeptide based on Aleuria aurantia lectin. Although all three assays showed a similar dynamic range, the microfluidic assays with fluorescent or SPR detection show an advantage in short analysis times. Furthermore, the microfluidic displacement assays provide a possibility to develop a one-step analytical platform.

     

A selective, noncovalent assay for base flipping in DNA

Base flipping, the conformational change of a nucleobase to an extrahelical position, is a key step in the enzymatic repair of damaged DNA. An assay that can detect the flipped-out species in free solution without covalent modification of the DNA would be desirable. The design and synthesis of a simple, sensitive, and rapid assay using specific noncovalent binding to pyrimidines by zinc-cyclen and a commonly used fluorescent reporter group, dansyl, is reported. The binding of the zinc-cyclen unit to a flipped-out thymine base results in a change in the fluorescent properties of the dansyl group that is distinct from nonspecific binding to duplex DNA or intercalation into either the flipped-in or flipped-out species. The assay was tested using fluorescence spectroscopy and detection at 533 +/- 5 nm with normal and abasic duplex DNA as negative and positive controls. The data obtained are fitted to a one-site binding model to determine the equilibrium constant for the two-step process involving base flipping and binding to be approximately 10-6 M.