A chemical tool for blue light-inducible proximity photo-crosslinking in live cells

Abstract

We developed a proximity photo-crosslinking method (<i>Spotlight</i>) with a 4-azido-<i>N</i>-ethyl-1,8-naphthalimide (AzNP) moiety that can be converted to reactive aryl nitrene species using ambient blue light-emitting diode light. Using an AzNP-conjugated HaloTag ligand (VL1), blue light-induced photo-crosslinked products of various HaloTag-conjugated proteins of interest were detected in subcellular spaces in live cells. Chemical or heat stress-induced dynamic changes in the proteome were also detected, and photo-crosslinking in the mouse brain tissue was enabled. Using <i>Spotlight</i>, we further identified the host interactome of SARS-CoV-2 nucleocapsid (N) protein, which is essential for viral genome assembly. Mass analysis of the VL1-crosslinked product of <i>N</i>-HaloTag in HEK293T cells showed that RNA-binding proteins in stress granules were exclusively enriched in the cross-linked samples. These results tell that our method can reveal the interactome of protein of interest within a short distance in live cells.