The development of reliable methods for the detection of minute amounts of antibodies directly in homogeneous solution represents one of the major tasks in the current research field of molecular diagnostics. We demonstrate the potential of fluorescence correlation spectroscopy (FCS) in combination with quenched peptide-based fluorescence probes for sensitive detection of p53 antibodies directly in homogeneous solution. Single tryptophan (Trp) residues in the sequences of short, synthetic peptide epitopes of the human p53 protein efficiently quench the fluorescence of an oxazine fluorophore attached to the amino terminal ends of the peptides. The fluorescence quenching mechanism is thought to be a photoinduced electron transfer reaction from Trp to the dye enabled by the formation of intramolecular complexes between dye and Trp. Specific recognition of the epitope by the antibody confines the conformational flexibility of the peptide. Consequently, complex formation between dye and Trp is abolished and fluorescence is recovered. Using fluorescence correlation spectroscopy (FCS), antibody binding can be monitored observing two parameters simultaneously: the diffusional mobility of the peptide as well as the quenching amplitude induced by the conformational flexibility of the peptide change significantly upon antibody binding. Our data demonstrate that FCS in combination with fluorescence-quenched peptide epitopes opens new possibilities for the reliable detection of antibody binding events in homogeneous solution.
We demonstrate how photoinduced electron transfer (PET) reactions can be successfully applied to monitor conformational dynamics in individual biopolymers. We present data about structural changes of single biomolecules like peptides by using quenching electron transfer reactions between tryptophan residues in close proximity to fluorescent dyes. Our results demonstrate that selective PET-reactions between fluorophores and amino acids represent a versatile tool to measure small-scale conformational dynamics in biopolymers on a wide range of time scales, extending from nanoseconds to seconds, at the single-molecule level under equilibrium conditions. That is, the monitoring of conformational dynamics of biopolymers with temporal resolutions comparable to those within reach using new techniques of molecular dynamic simulations. Furthermore, we demonstrate that the strong distance dependence of charge separation reactions on the sub-nanometer scale can be used to develop conformationally flexible PET-biosensors. These sensors enable the detection of specific target molecules in the sub-picomolar range and allow one to follow their molecular binding dynamics with temporal resolution.
We demonstrate how photoinduced electron transfer (PET) reactions can be successfully applied to monitor conformational dynamics in individual biopolymers. Single-pair fluorescence resonance energy transfer (FRET) experiments are ideally suited to study conformational dynamics occurring on the nanometer scale, e.g. during protein folding or unfolding. In contrast, conformational dynamics with functional significance, for example occurring in enzymes at work, often appear on much smaller spatial scales of up to several Angströms. Our results demonstrate that selective PET-reactions between fluorophores and amino acids or DNA nucleotides represent a versatile tool to measure small-scale conformational dynamics in biopolymers on a wide range of time scales, extending from nanoseconds to seconds, at the single-molecule level under equilibrium conditions. That is, the monitoring of conformational dynamics of biopolymers with temporal resolutions comparable to those within reach using new techniques of molecular dynamic simulations. We present data about structural changes of single biomolecules like DNA hairpins and peptides by using quenching electron transfer reactions between guanosine or tryptophan residues in close proximity to fluorescent dyes. Furthermore, we demonstrate that the strong distance dependence of charge separation reactions on the sub-nanometer scale can be used to develop conformationally flexible PET-biosensors. These sensors enable the detection of specific target molecules in the sub-picomolar range and allow one to follow their molecular binding dynamics with temporal resolution.
Circulating p53 autoantibodies are found to be a universal and highly specific tumor marker for malignant diseases. Hence, sereological screening for p53 autoantibodies at low concentration levels has become increasingly relevant for early-stage and follow-up of tumor diagnostics. We developed a new method for the highly sensitive detection of p53 antibodies in a homogeneous fluorescence assay format. Short, linear peptide derived form antibody recognition sequences so human p53 were labeled with an oxazine dye. Hydrophobic interactions constrain a conformation, where the dye interacts selectively with a tryptophan residue in the peptide sequence. Subsequently, the fluorescence of the dye is quenched efficiently due to electron transfer from the indole derivative to the dye in the excited state. Specific antibody recognition induces a conformational change in the peptide structure, repealing the dye-tryptophan interaction. Consequently, a fluorescence increase upon antibody binding signals the binding event. The long-wavelength absorption and emission characteristics of the probe and the use of a red pulsed diode laser as excitation source in a confocal fluorescence microscopic set-up allows ultra sensitive antibody detection at the single-molecule level. The effectiveness of the probes are highlighted by the detection of individual p53 autoantibodies directly in serum dilutions of cancer patients.
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