Cholera toxin entry into mammalian cells is mediated by binding of the pentameric B subunit (CTB) to ganglioside GM(1) in the cell membrane. We used flow cytometry to quantitatively measure in real time the interactions of fluorescently labeled pentameric cholera toxin B-subunit (FITC-CTB) with its ganglioside receptor on microsphere-supported phospholipid membranes. A model that describes the multiple steps of this mode of recognition was developed to guide our flow cytometric experiments and extract relevant equilibrium and kinetic rate constants. In contrast to previous studies, our approach takes into account receptor cross-linking, an important feature for multivalent interactions. From equilibrium measurements, we determined an equilibrium binding constant for a single subunit of FITC-CTB binding monovalently to GM(1) presented in bilayers of approximately 8 x 10(7) M(-1) while that for binding to soluble GM(1)-pentasaccharide was found to be approximately 4 x 10(6) M(-1). From kinetic measurements, we determined the rate constant for dissociation of a single site of FITC-CTB from microsphere-supported bilayers to be (3.21 +/- 0.03) x 10(-3) s(-1), and the rate of association of a site on FITC-CTB in solution to a GM(1) in the bilayer to be (2.8 +/- 0.4) x 10(4) M(-1) s(-1). These values yield a lower estimate for the equilibrium binding constant of approximately 1 x 10(7) M(-1). We determined the equilibrium surface cross-linking constant [(1.1 +/- 0.1) x 10(-12) cm(2)] and from this value and the value for the rate constant for dissociation derived a value of approximately 3.5 x 10(-15) cm(2) s(-1) for the forward rate constant for cross-linking. We also compared the interaction of the receptor binding B-subunit with that of the whole toxin (A- and B-subunits). Our results show that the whole toxin binds with approximately 100-fold higher avidity than the pentameric B-subunit alone which is most likely due to the additional interaction of the A(2)-subunit with the membrane surface. Interaction of cholera toxin B-subunit and whole cholera toxin with gangliosides other than GM(1) revealed specific binding only to GD1(b) and asialo-GM(1). These interactions, however, are marked by low avidity and require high receptor concentrations to be observed.

New genome sequence information is rapidly increasing the number of nucleic acid (NA) targets of use for characterizing and treating diseases. Detection of these targets by fluorescence-based assays is often limited by fluorescence background from unincorporated or unbound probes that are present in large excess over the target. To solve this problem, energy transfer-based probes have been developed and used to reduce the fluorescence from unbound probes. Although these probes have revolutionized NA target detection, their use requires scrupulous attention to design constraints, extensive probe quality control, and individually optimized experimental conditions. Here, we describe a simpler background reduction approach using singly labeled quencher oligomers to suppress excess unbound probe fluorescence following probe-target hybridization. A second limitation of most fluorescence-based NA target detection and quantification assays is the requirement for enzymatic amplification of target or signal for sensitivity. Amplification steps make quantification of original target copy number problematic because of variations in amplification efficiencies between the sequence targets and the experimental conditions. To avoid amplification, we coupled our quenching approach to a two-color NA assay with correlated, two-color, single-molecule fluorescence detection. We demonstrate a >100-fold background reduction and detection of targets present at concentrations as low as 100 fM using the two-color assay. The application of this technique to the detection and quantification of specific mRNA sequences enabled us to estimate beta-actin copy numbers in cell-derived total RNA without an amplification step.

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In the use of non-antibody proteins as affinity reagents, diversity has generally been derived from oligonucleotide-encoded random amino acids. Although specific binders of high-affinity have been selected from such libraries, random oligonucleotides often encode stop codons and amino acid combinations that affect protein folding. Recently it has been shown that specific antibody binding loops grafted into heterologous proteins can confer the specific antibody binding activity to the created chimeric protein. In this paper, we examine the use of such antibody binding loops as diversity elements. We first show that we are able to graft a lysozyme-binding antibody loop into green fluorescent protein (GFP), creating a fluorescent protein with lysozyme-binding activity. Subsequently we have developed a PCR method to harvest random binding loops from antibodies and insert them at predefined sites in any protein, using GFP as an example. The majority of such GFP chimeras remain fluorescent, indicating that binding loops do not disrupt folding. This method can be adapted to the creation of other nucleic acid libraries where diversity is flanked by regions of relative sequence conservation, and its availability sets the stage for the use of antibody loop libraries as diversity elements for selection experiments.

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Recently developed single-molecule spectroscopy (SMS) permits the analysis of fluorescent mixtures one molecule at a time. SMS methods provide the means to make rapid measurements on small, complex samples without the need for separations and target amplification enabling a new class of ultrasensitive nucleic acid assays. Here we give a brief overview of the current state of the art of SMS nucleic acid analysis and discuss ongoing work in our laboratory on two-color single-molecule fluorescence detection of specific nucleic acid sequences. In the future, two-color SMS nucleic acid assays will be used for a variety of applications including: gene expression analysis, disease detection and genomics.

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