Development of a Method To Prioritize Protein-Ligand Pairs on Beads Using Protein Conjugated to a Near-IR Dye

The development of techniques to screen one-bead-one-compound (OBOC) libraries remains a critical step in identifying small molecules that bind target proteins. While great strides have been made, there remains a need to continue to develop OBOC screening techniques that not only reliably identify hit molecules but also can distinguish poor from excellent binders in a single screen. Similarly, relatively strong binding between a small molecule and protein target is required to be considered a hit from the initial pool of screened molecules. Here, we present the framework for a method to screen OBOC libraries using proteins and antibodies stained with a near-infrared (NIR)-emitting fluorophore. These labeled proteins provide significant signal at very low concentrations because of their fluorescence quantum yield. This work revealed that we can detect proteins and antibodies interacting with a known binding partner at low nanomolar concentrations; binding is specific, and known binders to carbonic anhydrase can be detected and ranked.