Publication details
Influence of the geometry of fluorescently labelled DNA constructs on fluorescence anisotropy assay
| Authors | |
|---|---|
| Year of publication | 2020 |
| Type | Article in Periodical |
| Magazine / Source | Biochemical and biophysical research communications |
| MU Faculty or unit | |
| Citation | |
| Web | https://www.sciencedirect.com/science/article/pii/S0006291X2030749X |
| Doi | http://dx.doi.org/10.1016/j.bbrc.2020.04.025 |
| Keywords | Fluorescence anisotropy; DNA-Encoded chemical library; Drug discovery; Hit validation; Small molecules |
| Attached files | |
| Description | DNA-encoded chemical libraries (DECLs) are powerful tools for modern drug discovery. A DECL is apooled mixture of small molecule compounds, each of which is tagged with a unique DNA sequencewhich 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 arebinding the target. While the DECL technology itself is ultra-high throughput, the following re-synthesisof identified compounds for orthogonal validation experiments remains the bottleneck. Using existingDNA-small molecule conjugates directly for affinity measurements, as opposed to complete compoundresynthesis, could accelerate the discovery process. To this end, we have tested various geometries offluorescently-labelled DNA constructs forfluorescence anisotropy (FA) experiments. Minimizing thedistance between thefluorescent moiety and ligand can maximize the correlation between ligand-protein interaction and corresponding change influorophore rotational freedom, thus leading tolarger, easier to interpret changes in FA values. However, close proximity can also cause artifacts due topotentially promiscuous interactions betweenfluorophore and protein. By balancing these two oppositeeffects, we have identified applicablefluorescently labelled DNA constructs displaying either a singleligand or pairs of fragments for affinity measurement using a FA assay. |
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