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

Pratyush Kumar Mishra; Myeong Gyun Kang; Hakbong Lee; Seungjoon Kim; Subin Choi; Nirmali Sharma; Cheol Min Park; Jaewon Ko; Changwook Lee; Jeong Kon Seo; Hyun Woo Rhee

doi:10.1039/d1sc04871f

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

Pratyush Kumar Mishra, Myeong Gyun Kang, Hakbong Lee, Seungjoon Kim, Subin Choi, Nirmali Sharma, Cheol Min Park, Jaewon Ko, Changwook Lee, Jeong Kon Seo, Hyun Woo Rhee

Department of Brain Sciences

Research output: Contribution to journal › Article › peer-review

13 Scopus citations

Abstract

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.

Original language	English
Pages (from-to)	955-966
Number of pages	12
Journal	Chemical Science
Volume	13
Issue number	4
DOIs	https://doi.org/10.1039/d1sc04871f
State	Published - 28 Jan 2022

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© 2022 The Royal Society of Chemistry.

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title = "A chemical tool for blue light-inducible proximity photo-crosslinking in live cells",

abstract = "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.",

author = "Mishra, {Pratyush Kumar} and Kang, {Myeong Gyun} and Hakbong Lee and Seungjoon Kim and Subin Choi and Nirmali Sharma and Park, {Cheol Min} and Jaewon Ko and Changwook Lee and Seo, {Jeong Kon} and Rhee, {Hyun Woo}",

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AU - Mishra, Pratyush Kumar

AU - Kang, Myeong Gyun

AU - Lee, Hakbong

AU - Kim, Seungjoon

AU - Choi, Subin

AU - Sharma, Nirmali

AU - Park, Cheol Min

AU - Ko, Jaewon

AU - Lee, Changwook

AU - Seo, Jeong Kon

AU - Rhee, Hyun Woo

PY - 2022/1/28

Y1 - 2022/1/28

N2 - 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.

AB - 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.

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DO - 10.1039/d1sc04871f

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VL - 13

SP - 955

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JO - Chemical Science

JF - Chemical Science

IS - 4

ER -

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

Abstract

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