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Abstract
We developed novel assays for high-throughput detection of one or many kinases or proteases. The assays use hundreds of different peptide substrates, each covalently linked to an oligonucleotide tag. After incubation with sample, the pool of substrates is hybridized to a microarray containing oligonucleotides complementary to the tag sequences. We screened several specific chemistries for the conjugation based on the following criteria: easy derivatization of oligonucleotides and peptides; high efficiency of the conjugation reaction; good stability of the conjugates; and satisfactory conjugate performance in our assays. We have validated selected method during the successful generation of thousands oligonucleotide-peptide conjugates.
The protease project was partially funded by NIH grant R44 AI056869-02; the kinase project was partially funded by NIH grant R43 GM071272-01A1. We thank Melissa Shults, Nicholas Nelson, Joe Musmacker, Anu Srinivasan, Veronica Shevchenko, Bahram Kermani, and Gali Steinberg-Tatman for their help with this project.