First Fluorescence Imaging of Organisms Living at 75°C Using a Fully Designed Tag With No Natural Protein

Fluorescence imaging of microorganisms living at 75°C has become possible for the first time. An artificial fluorescent tag designed from scratch without using natural proteins has been developed.

A research team led by Hwangbo at the University of California, San Francisco (UCSF), announced on the 1st (local time) in the international journal Cell that it had developed Rhobin, a fully designed protein that binds the rhodamine dye. The first author is researcher Tianyu.

Existing fluorescent tools have been made by modifying natural proteins, such as green fluorescent protein (GFP) and HaloTag. As a result, fluorescent proteins matured slowly and had a narrow range of colors, while HaloTag was large and heat-sensitive.

The research team set aside natural proteins and computationally designed a protein from scratch to target the shared structure of rhodamine dyes. Rhodamine shifts between an internally esterified (L) form, which is nonfluorescent, and a strongly fluorescent zwitterionic (Z) form; the designed protein turns on fluorescence by regulating this balance.

Its performance also surpassed that of existing tools. Rhobin is much smaller and brighter in mammalian cells. Its reversible binding makes it suitable for super-resolution STED imaging and long-term single-molecule tracking in living cells, where it performed better than HaloTag.

In particular, its heat resistance enabled the first fluorescence imaging of living cells of the thermoacidophilic archaeon Sulfolobus, which lives in environments at 75°C. Existing tools could not access this area.

The design success rate was also high. Eight of the nine sequences tested showed strong binding. The research team said Rhobin could serve as a modular platform for developing next-generation biosensors and multicolor imaging systems.

However, the current design focuses on rhodamine dyes, and its extension to other dyes requires further validation. The success rate is not 100%, the computational cost of design is substantial, and long-term stability and biocompatibility in complex biological tissues also require further evaluation.