Competitive Binding Study Revealing the Influence of Fluorophore Labels on Biomolecular Interactions

Abstract

Fluorescence methods are important tools in modern biology. Direct labeling of biomolecules with a fluorophore might, however, change interaction surfaces. Here, we introduce a competitive binding assay in combination with fluorescence correlation spectroscopy that reports binding affinities of both labeled and unlabeled biomolecules to their binding target. We investigated how fluorophore labels at different positions of a DNA oligonucleotide affect hybridization to a complementary oligonucleotide and found dissociation constants varying within 2 orders of magnitude. We next demonstrated that placing a fluorophore label at position Leu280 in the protein ligand internalin B does not alter the binding affinity to the MET receptor tyrosine kinase, compared to unlabeled internalin B. Our approach is simple to implement and can be applied to investigate the influence of fluorophore labels in a large variety of biomolecular interactions.

A multiparametric fluorescence assay for screening aptamer–protein interactions based on microbeads

“…We uniformly labelled the aptamers with the water-soluble fluorescent dye sulfo-Cy5, which is a commonly used label in bioanalytics (Table 1 ). It should be stressed that any modification can change the properties of aptamers, especially the three-dimensional structure 13 , 26 . The solubility of an aptamer can be increased or decreased depending upon the hydrophilicity of the label.…”

PharmacoSTORM nanoscale pharmacology reveals cariprazine binding on Islands of Calleja granule cells

“…Functional pharmacological assays are convenient for the rapid screening of the best candidates, as we demonstrated via the development of a fluorescent ligand for the CB 1 cannabinoid receptor, the MAGL enzyme and a labeled analog of an approved drug (fluo-cariprazine). The exact effects of fluorophore tagging on equilibrium or kinetic ligand-binding parameters can certainly be obtained by further radioactivity- or fluorescence-based methods 55 , 56 . Moreover, the specificity of target binding can be analyzed by appropriate control PharmacoSTORM experiments using untransfected cells, displacement assays, point-mutant receptors for binding sites, or tissues obtained from knockout mice as we demonstrated all four control approaches in the case of fluo-cariprazine.…”

Comparing Transient Oligonucleotide Hybridization Kinetics Using DNA-PAINT and Optoplasmonic Single-Molecule Sensing on Gold Nanorods

“…[18][19][20] Despite this steady progress, single-molecule techniques suffer from different drawbacks that limit the information one can extract. For example, it is well-known that fluorescent labels may affect the kinetics and dynamics of biomolecular systems, [21][22][23] while LSPR-based approaches use local field enhancements that are not uniform across a plasmonic nanostructure, and the local surface geometry and heterogeneity of plasmonic nanoparticles can play a significant role in the observed statistics. [24][25][26] In view of this, finding an accurate description of bioprocesses at the singlemolecule level will require the combination of information arising from different methods applied to the same molecular system.…”