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Influence of the geometry of fluorescently labelled DNA constructs on fluorescence anisotropy assay
T. Brom, FV. Reddavide, S. Heiden, M. Thompson, Y. Zhang
Language English Country United States
Document type Journal Article, Research Support, Non-U.S. Gov't
- MeSH
- DNA chemical synthesis chemistry MeSH
- Fluorescent Dyes chemical synthesis chemistry MeSH
- Fluorescence Polarization MeSH
- Small Molecule Libraries chemical synthesis chemistry pharmacology MeSH
- Ligands MeSH
- Drug Discovery MeSH
- Drug Evaluation, Preclinical MeSH
- Combinatorial Chemistry Techniques MeSH
- Binding Sites MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
DNA-encoded chemical libraries (DECLs) are powerful tools for modern drug discovery. A DECL is a pooled mixture of small molecule compounds, each of which is tagged with a unique DNA sequence which functions as a barcode. After incubation with a drug target and washing to remove non-binders, the bound molecules are eluted and submitted for DNA sequencing to determine which molecules are binding the target. While the DECL technology itself is ultra-high throughput, the following re-synthesis of identified compounds for orthogonal validation experiments remains the bottleneck. Using existing DNA-small molecule conjugates directly for affinity measurements, as opposed to complete compound resynthesis, could accelerate the discovery process. To this end, we have tested various geometries of fluorescently-labelled DNA constructs for fluorescence anisotropy (FA) experiments. Minimizing the distance between the fluorescent moiety and ligand can maximize the correlation between ligand-protein interaction and corresponding change in fluorophore rotational freedom, thus leading to larger, easier to interpret changes in FA values. However, close proximity can also cause artifacts due to potentially promiscuous interactions between fluorophore and protein. By balancing these two opposite effects, we have identified applicable fluorescently labelled DNA constructs displaying either a single ligand or pairs of fragments for affinity measurement using a FA assay.
References provided by Crossref.org
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