A STING-based biosensor affords broad cyclic dinucleotide detection within single living eukaryotic cells.

Nature Communications
Alex J PollockJoshua J Woodward

Abstract

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.

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Citations

Nov 4, 2020·Chembiochem : a European Journal of Chemical Biology·Shivam A ZaverJoshua J Woodward
Nov 7, 2020·EMBO Reports·Jonny Hertzog, Jan Rehwinkel

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Methods Mentioned

BETA
biosensors
FRET
biosensor
flow cytometry
transfection
flow
gel filtration
FACS
Protein Assay

Software Mentioned

BioSTINGʼs
PyMol
STING
CTD
BioSTING
Prism
DRaCALA

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