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

• Published in: Nature communications Vol. 11; no. 1; pp. 3533 - 13
• Main Authors: Pollock, Alex J., Zaver, Shivam A., Woodward, Joshua J.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 15.07.2020; Nature Publishing Group; Nature Portfolio
• Subjects: 13/31; 14/33; 14/35; 14/63; 631/1647/1888; 631/1647/527/2047; 631/250; 631/45; 82/80; 82/83; 9/10; Animals; Autoimmune diseases; Bacillus subtilis - chemistry; Bacterial diseases; Biosensing Techniques; Biosensors; Cyclic GMP - metabolism; Domains; Energy transfer; Engineers; Eukaryotes; Flow Cytometry; Fluorescence Resonance Energy Transfer; HEK293 Cells; Humanities and Social Sciences; Humans; Immunity, Innate; Innate immunity; Ligands; Membrane Proteins - chemistry; Mice; multidisciplinary; Mutagenesis; Nucleotides; Nucleotides, Cyclic - analysis; Pathological effects; Protein Domains; Protein Multimerization; Real time; Recombinant Proteins; Resonance; Science; Science (multidisciplinary); Second messengers; Signal Transduction
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-020-17228-y

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. Cyclic dinucleotides are conserved second messengers but current detection methods are limited. Here the authors engineer a Förster resonance energy transfer (FRET) based biosensor, BioSTING, which gives real-time in vitro detection of these nucleotides.