Described herein is an application of the copper(I)-catalyzed Huisgen [3+2] cycloaddition between azides and alkynes,
or click chemistry, for the universal two-step detection of biological macromolecules. The first step involves the
metabolic incorporation of an azide or alkyne probe into a macromolecule of interest. The second step involves the
click chemistry conjugation of the labeled macromolecule with a partner alkyne or azide-reactive fluorescent probe to
form a stable triazole ring conjugate. The fluorescently tagged molecules can be subsequently detected by a number of
different fluorescent readout platforms including flow cytometry, fluorescence imaging, and 1-D/2-D gel imaging. We
demonstrate application of this technology in two different labeling schemes. First, the labeling of newly synthesized
DNA in a novel cell proliferation assay, and second, in the labeling of specific glycoprotein subclasses for biomarker
discovery applications. In each case, azide or alkyne probes are introduced metabolically with subsequent detection
using click chemistry. Utilization of the cellular enzymatic machinery for high-fidelity target molecule labeling
combined with the superior efficiency of click chemistry detection results in highly versatile macromolecular labeling
platforms that are unmatched in sensitivity and selectivity.
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