The smallest near-infrared fluorescence complementation system for imaging protein-protein and RNA-protein interactions.

ABSTRACT

Bimolecular fluorescence complementation (BiFC) and its derivative molecular biosensor systems provide effective tools for visualizing biomolecular interactions. The introduction of red and near-infrared fluorescence emission proteins has expanded the spectrum of signal generating modules, enabling BiFC for in vivo imaging. However, the large size of the signal module of BiFC can hinder the interaction between proteins under investigation. In this study, we constructed the near-infrared BiFC and TriFC systems by splitting miRFP670nano, the smallest cyanobacteriochrome-evolved phytochrome available. The miRFP670nano-BiFC sensor system identified and enabled visualization of protein-protein interactions in living cells and live mice, and afforded a faster maturation rate and higher photostability and cellular stability when compared with those of reported near-infrared BiFC systems. We used the miRFP670nano-BiFC sensor system to identify interactions between the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and cellular stress granule proteins in living cells and found that the N protein downregulated the expression level of granule protein G3BP1. With the advantages of small size and long wavelength emission of the signal module, the proposed molecular biosensor system should be suitable for various applications in cell imaging studies.