Abstract
Fluorescent drug screening assays are essential for tyrosine kinase inhibitor discovery. Here we demonstrate a flexible, antibody-free TR-LRET kinase assay strategy that is enabled by the combination of streptavidin-coated quantum dot (QD) acceptors and biotinylated, Tb3+ sensitizing peptide donors. By exploiting the spectral features of Tb3+ and QD, and the high binding affinity of the streptavidin-biotin interaction, we achieved multiplexed detection of kinase activity in a modular fashion without requiring additional covalent labeling of each peptide substrate. This strategy is compatible with high-throughput screening, and should be adaptable to the rapidly changing workflows and targets involved in kinase inhibitor discovery.
Highlights
Protein tyrosine kinases have been significant drug targets for decades, and an ever-growing number of compounds are being tested against various kinases for their therapeutic potential
We have explored the design and application of such sequences[25] for novel time-resolved luminescence kinase assays in TRL and TR-LRET forms[20,27] for a variety of kinases involved in cancer signaling, including a dual-plexed approach using small molecule fluorophores to differentiate between substrates[27]
Because of the broad and continuous absorption spectra of quantum dot (QD), which are highest in the UV to short wavelength visible range regardless of emission color (Fig. 1a), the luminescence emission from Tb3+ is efficiently exploited and provides more flexible LRET pair options (Fig. 1a) than conventional organic fluorophores
Summary
Protein tyrosine kinases have been significant drug targets for decades, and an ever-growing number of compounds are being tested against various kinases for their therapeutic potential. We report a more flexible strategy for a multiplexed, antibody-free kinase assay using TR-LRET between quantum dot (QD) fluorophores and phosphorylation-dependent lanthanide-sensitizing peptide biosensors.
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