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

We developed a proximity photo-crosslinking method (Spotlight) with a 4-azido-N-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-crosslin...

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Detalles Bibliográficos
Autores principales: Mishra, Pratyush Kumar, Kang, Myeong-Gyun, Lee, Hakbong, Kim, Seungjoon, Choi, Subin, Sharma, Nirmali, Park, Cheol-Min, Ko, Jaewon, Lee, Changwook, Seo, Jeong Kon, Rhee, Hyun-Woo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Royal Society of Chemistry 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8790779/
https://www.ncbi.nlm.nih.gov/pubmed/35211260
http://dx.doi.org/10.1039/d1sc04871f
Descripción
Sumario:We developed a proximity photo-crosslinking method (Spotlight) with a 4-azido-N-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 Spotlight, 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 N-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.