Antibody-Mediated Fluorescence Enhancement Based on Shifting the Intramolecular Dimer .dblarw. Monomer Equilibrium of Fluorescent Dyes

Abstract

A novel concept is described for directly coupling fluorescence emission to protein-ligand binding. It is based on shifting the intramolecular monomer<-->dimer equilibrium of two fluorescent dyes linked by a short spacer. A 13-residue peptide, recognized by a monoclonal antibody against human chorionic gonadotrophin (hCG), was labeled with fluorescein (F) and tetramethylrhodamine (T) at its N- and C-terminus, respectively. Spectral evidence suggests that when the conjugate is free in solution, F and T exist as an intramolecular dimer. Fluorescence quenching of fluorescein and rhodamine is approximately 98% and approximately 90%, respectively, due to dimerization. When the double-labeled peptide is bound to anti-hCG, however, the rhodamine fluorescence increases by up to 7.8-fold, depending upon the excitation wavelength. This is attributed to the dissociation of intramolecular dimers brought about by conformational changes of the conjugate upon binding. Fluorescein fluorescence, on the other hand, was still quenched because of excited-state energy transfer and residual ground-state interactions. Antibody binding also resulted in a approximately 3.4-fold increase in fluorescence anisotropy of the peptide. These changes in intensity and anisotropy allow direct measurement of antigen-antibody binding with a fluorescence plate reader or a polarization analyzer, without the need for separation steps and labeling antibodies. Because recent advances in peptide technology have allowed rapid and economical identification of antigen-mimicking peptides, the double-labeled peptide approach offers many opportunities for developing new diagnostic assays and screening new therapeutic drugs. It also has many potential applications to techniques involving recombinant antibodies, biosensors, cell sorting, and DNA probes.

Intramolecular Dimers: A New Design Strategy for Fluorescence‐Quenched Probes

“…[25] Other studies have noted that efficient quenching can occur despite the fact that the absorption band of the quencher does not overlap with the emission band of the fluorophore-this indicates that a mechanism besides FRET occurs. [26,27] Packard and co-workers have systematically investigated some aspects of quenching in homo-dual-labeled peptides. They describe intramolecular homodimers in profluorescent protease substrates, structural characteristics of fluorophores that promote homodimer formation (surprisingly, hydrophobicity was not very important), [28] and how length and conformation of the peptide plus linker controls dimer formation.…”

“…Labels Assay Fluorescence increase [28] NorFES rhodamine ± serine 10:1 undecapeptide rhodamine protease [27] decapeptide rhodamine ± malarial 30:1 rhodamine protease [26] 13-residue fluorescein ± monoclonal 8:1 increase peptide rhodamine antibody for rhodamine [30] octapeptide rhodamine± BHQ2 Trypsin 100:1…”

Mechanism‐Based Fluorescent Reporter for Protein Kinase A Detection

“…[18] The intrinsic fluorescence of fluorescein and rhodamine in this conjugate was 64-fold and tenfold lower than that of the monolabeled peptide, respectively. This higher quenching of fluorescein was explained as the result of both dimer formation (static process) and the excited-state energy transfer to rhodamine (dynamic process).…”

“…This higher quenching of fluorescein was explained as the result of both dimer formation (static process) and the excited-state energy transfer to rhodamine (dynamic process). [18] However, the significant decrease in fluorescence emission of TAMRA suggests that Fçrster fluorescence resonance energy transfer (FRET) is not the predominant mechanism in 4 R.…”

A Target Cell–Specific Activatable Fluorescence Probe for In vivo Molecular Imaging of Cancer Based on a Self-Quenched Avidin-Rhodamine Conjugate

“…Increased fluorescence signal after exposure to SDS was also observed with Av-0.5ROX. The precise mechanism of the fluorescence enhancement and reduction in rhodamine dyes is not yet understood (15)(16)(17)(18)(19), and the reason why SDS can amplify the signal of Av-0.5ROX and Av-3ROX in general is still unclear. We suggest that the detergent function of SDS can change the conformation of the avidin chain, which allows the rhodamine molecules to escape from the highly charged peptide, which normally compromises the fluorescence signal of rhodamine X.…”