Abstract
Here, we developed nanobody-assisted nanoluciferase fragment complementation for in situ measurement and visualization of endogenous protein-protein interaction (NanaPPI). When an interaction occurs, primary antibodies for two proteins bring the proximity of secondary nanobody-fused small/large fragment to reassemble into an intact NanoLuc variant, thus transforming interaction events to luminescent signals in situ with high sensitivity. Compared to proximity ligation assay, NanaPPI has a similar signal-to-background ratio, but it is more convenient with faster procedures, easier readout and lower cost. NanaPPI not only allows direct detection of low abundant interactions, but also visualizes protein-protein interaction events in fixed cells and tissue sections. By applying NanaPPI, disruption of PPIs by inhibitors and distinct PPI levels under physiological or pathological conditions, can be quantified efficiently. Unknown interactions YTHDF2/G3BP1 and RNA m6A/G3BP1 can be also identified by NanaPPI under unstressed conditions, with drastic increasing under arsenite stress. The interaction between RNA m6A and G3BP1 is largely reduced upon YTHDF2 knockdown, indicating YTHDF2 mediates the enrichment of m6A-modified mRNA in stress granules. In conclusion, NanaPPI provides a robust, easy, and economical method for rapid in situ measurement of PPIs in cells and tissues, which has great potential for new PPI identification, PPI inhibitor screening, and PPI biomarker-based diagnosis in clinics.
Title
Nanobody-assisted nanoluciferase fragment complementation for in situ measurement and visualization of endogenous protein-protein interaction
Authors
Qianqian Li, Huijuan Liu, Xiangjun Du, Yafan Xie, Yanwei Chen, Juhui Qiu, Yiqin Gao, Qin Peng
Journal Information
Biosensors and Bioelectronics (2025)
DOI
https://doi.org/10.1016/j.bios.2024.117102
