AI-designed proteins for imaging inside living cells
Cells are like metropolises, home to millions of molecular residents. If one were to stand atop a high-rise, trying to identify most of its inhabitants would seem an impossible task. Even with the sophisticated imaging tools currently available to scientists, it is challenging to zoom in on specific molecules and view them with a high degree of detail.
To address this limitation, researchers have developed a new class of synthetic fluorescent protein tags, called NovoTags. The collaboration culminated in a paper, now published in Science, describing how versatile NovoTags can help locate specific proteins and probe their interactions inside human cells using an array of advanced light microscopy techniques, including super-resolution fluorescence light microscopy.
The resarchers first developed the NovoTag binders against three Janelia Fluor (JF) dyes spanning the visible spectrum, complementing existing systems such as HaloTag and SNAP-tag and expanding the possibilities for multicolour imaging. These binders are small, de novo designed proteins that can be genetically 'tagged’ to proteins of interest and that can bind specific dyes, making them visible using fluorescence microscopy techniques.
“This study shows that designed proteins can modulate a fluorophore’s photophysical properties, offering new insight into design principles that govern photophysical function in protein-fluorophore complexes,” said the author.
The authors then developed a split, inducible version, named NovoSplit, engineering a molecular switch that only assembles once its target dye is present. "A central aspect of the NovoSplit system is inducibility," the author pointed out. "We wanted to control protein-protein interactions, so we needed a chemically inducible system." Essentially, this allows scientists to ensure that two proteins being studied only come in close contact with each other when an external agent – a dye – is added to the cell environment.
"Back at EMBL, I characterised the NovoTags and NovoSplit in human cell lines using advanced microscopy, particularly live STED microscopy and fluorescence lifetime imaging microscopy (FLIM)," said the author. "By combining spectral separation with fluorescence lifetime information, we can potentially distinguish many more labels than would be possible using spectral separation alone."
This means that in the future scientists could use this tagging system to simultaneously view or track up to 30 different proteins, by labelling each using a unique combination of emission spectrum (i.e. colour) and fluorescence lifetime (i.e. how long something emits light). The NovoTag sequences and complementary dyes are freely available to the scientific community.
In essence, the NovoSplit system works quite simply. The NovoTag is split into two halves, each linked to a different protein in a cell. When a specific JF dye is added to the system, it acts as a molecular glue that assembles the two parts, allowing scientists to control the interaction between the two proteins.
Further exciting applications are in store. Cryo-correlative light and electron microscopy (cryo-CLEM) is a cutting-edge imaging method where the same cell can be viewed under a light and an electron microscope in a fully native state. The resulting images can be overlaid on each other to yield information neither technique could achieve alone.
"Our NovoSplit system forms the basis for the development of inducible cryo-CLEM tags," said the author. Cryo-CLEM tags that are fluorescent and structurally distinctive enough to be identified directly inside cryo-electron tomograms would be a major leap for in-cell structural biology research.
It also reflects how building these tags depends on a modern protein design pipeline. “We use RFdiffusion to design protein backbones and LigandMPNN to generate amino acid sequences,” explained the author. “Candidate proteins are computationally filtered using AlphaFold and RoseTTAFold before being screened experimentally using yeast surface display, FACS, next-generation sequencing, fluorescence polarisation assays, and size-exclusion chromatography.”
"Ultimately, this work aims to expand the possibilities for identifying proteins directly within native cells by combining advanced fluorescence microscopy with cryo-electron tomography," the author explained. The possibilities such Cryo-CLEM tags could unlock are significant, enabling scientists to better study the busy alleyways of cells and their residents.
