A STING-based biosensor affords broad cyclic dinucleotide detection within single living eukaryotic cells.
Alex J PollockShivam A ZaverJoshua J WoodwardPublished in: Nature communications (2020)
Cyclic dinucleotides (CDNs) are second messengers conserved across all three domains of life. Within eukaryotes they mediate protective roles in innate immunity against malignant, viral, and bacterial disease, and exert pathological effects in autoimmune disorders. Despite their ubiquitous role in diverse biological contexts, CDN detection methods are limited. Here, using structure guided design of the murine STING CDN binding domain, we engineer a Förster resonance energy transfer (FRET) based biosensor deemed BioSTING. Recombinant BioSTING affords real-time detection of CDN synthase activity and inhibition. Expression of BioSTING in live human cells allows quantification of localized bacterial and eukaryotic CDN levels in single cells with low nanomolar sensitivity. These findings establish BioSTING as a powerful kinetic in vitro platform amenable to high throughput screens and as a broadly applicable cellular tool to interrogate the temporal and spatial dynamics of CDN signaling in a variety of infectious, malignant, and autoimmune contexts.
Keyphrases
- energy transfer
- quantum dots
- high throughput
- label free
- induced apoptosis
- loop mediated isothermal amplification
- cell cycle arrest
- sensitive detection
- real time pcr
- multiple sclerosis
- gold nanoparticles
- poor prognosis
- endoplasmic reticulum stress
- sars cov
- binding protein
- cell death
- oxidative stress
- signaling pathway
- gene expression
- genome wide
- drug induced
- single cell
- cell proliferation
- long non coding rna
- dna methylation
- cell free
- living cells