Adaptable, Turn-On Monobody (ATOM) Fluorescent Biosensors for Multiplexed Detection in Cells.
Harsimranjit SekhonJeung-Hoi HaMaria F PrestiSpencer B ProcopioPaige O MirskyAnna M JohnStewart N LohPublished in: bioRxiv : the preprint server for biology (2023)
A grand challenge in biosensor design is to develop a single molecule, fluorescent protein-based platform that can be easily adapted to recognize targets of choice. Conceptually, this can be achieved by fusing a small, antibody-like binding domain to a fluorescent protein in such a way that target binding activates fluorescence. Although this design is simple to envision, its execution is not obvious. Here, we created a family of adaptable, turn-on monobody (ATOM) biosensors consisting of a monobody, circularly permuted at one of two positions, inserted into a fluorescent protein at one of three surface loops. Multiplexed imaging of live human cells co-expressing cyan, yellow, and red ATOM sensors detected the biosensor targets (WDR5, SH2, and hRAS proteins) that were localized to the nucleus, cytoplasm, and plasma membrane, respectively, with high specificity. ER- and mitochondria-localized ATOM sensors also detected ligands that were targeted to those organelles. Fluorescence activation involved ligand-dependent chromophore maturation with fluorescence turn-on ratios of >20-fold in cells and up to 100-fold in vitro . The sensing mechanism was validated with three arbitrarily chosen monobodies inserted into jellyfish as well as anemone lineages of fluorescent proteins, suggesting that ATOM sensors with different binding specificities and additional colors can be generated relatively quickly.
Keyphrases
- living cells
- single molecule
- label free
- quantum dots
- fluorescent probe
- molecular dynamics
- induced apoptosis
- sensitive detection
- binding protein
- atomic force microscopy
- cell cycle arrest
- electron transfer
- protein protein
- low cost
- gold nanoparticles
- high resolution
- energy transfer
- single cell
- dna binding
- cell death
- endoplasmic reticulum stress
- oxidative stress
- transcription factor
- reactive oxygen species
- high throughput
- small molecule
- loop mediated isothermal amplification