高亮度近红外 BRET 系统用于活细胞中分子事件的成像。

Highly Bright NIR-BRET System for Imaging Molecular Events in Live Cells.

机构信息

Department of Applied Chemistry, Faculty of Science and Technology, Graduate School of Science and Technology, Keio University, Yokohama, Kanagawa, Japan.

Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.

出版信息

Methods Mol Biol. 2021;2274:247-259. doi: 10.1007/978-1-0716-1258-3_21.

DOI:10.1007/978-1-0716-1258-3_21

PMID:34050477

Abstract

The present protocol demonstrates a novel mammalian cell imaging platform exerting a bioluminescence resonance energy transfer (BRET) system. This platform achieves a ~300 nm blue-to-near infrared shift of the emission (NIR-BRET) with the development of a unique coelenterazine (CTZ) derivative named BBlue2.3 and a fusion reporter protein probe named iRFP-RLuc8.6-535SG. The best NIR-BRET shift was achieved by tuning the blue emission peak of BBlue2.3 to a Soret band of the iRFP. In mammalian cells, BBlue2.3 emits light that is ~50-fold brighter than DeepBlueC in cell imaging when combined with RLuc8.6-535SG. This NIR-BRET platform is sufficiently brighter to be used for imaging live mammalian cells at single-cell level, and also for imaging metastases in deep tissues in live mice without generating considerable autoluminescence. This unique optical platform provides the brightest NIR-BLI template that can be used for imaging a diverse group of cellular events in living subjects.

摘要

本方案展示了一种新型的哺乳动物细胞成像平台，该平台采用了生物发光共振能量转移（BRET）系统。通过开发一种名为 BBlue2.3 的独特腔肠素（CTZ）衍生物和一种名为 iRFP-RLuc8.6-535SG 的融合报告蛋白探针，实现了约 300nm 的蓝移至近红外（NIR-BRET）。通过将 BBlue2.3 的蓝移发射峰调谐到 iRFP 的 Soret 带，实现了最佳的 NIR-BRET 位移。在哺乳动物细胞中，BBlue2.3 与 RLuc8.6-535SG 结合时，在细胞成像中发射的光比 DeepBlueC 亮约 50 倍。该 NIR-BRET 平台足够亮，可以用于在单细胞水平上对活哺乳动物细胞进行成像，也可以用于在活小鼠的深部组织中对转移进行成像，而不会产生可观的自发荧光。这个独特的光学平台提供了最亮的 NIR-BLI 模板，可用于在活体中对多种细胞事件进行成像。