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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Immunology

Background:

  • Cyclic dinucleotides (CDNs) are conserved second messengers crucial for innate immunity and implicated in autoimmune diseases.
  • Current methods for detecting CDNs are limited, hindering research into their diverse biological roles.
  • Understanding CDN signaling is vital for developing treatments for infectious, malignant, and autoimmune conditions.

Purpose of the Study:

  • To engineer a novel biosensor for sensitive and real-time detection of cyclic dinucleotides (CDNs).
  • To establish a platform for quantifying CDN levels in vitro and within live cells.
  • To enable interrogation of CDN dynamics in various disease contexts.

Main Methods:

  • Structure-guided design of the murine STING CDN binding domain.
  • Development of a Förster resonance energy transfer (FRET) based biosensor named BioSTING.
  • Expression of BioSTING in live human cells for intracellular CDN quantification.

Main Results:

  • Recombinant BioSTING enables real-time detection of CDN synthase activity and inhibition.
  • BioSTING allows quantification of localized CDN levels in single human cells with low nanomolar sensitivity.
  • The biosensor demonstrated applicability in interrogating CDN signaling in diverse cellular contexts.

Conclusions:

  • BioSTING is a powerful kinetic platform for in vitro high-throughput screening.
  • BioSTING serves as a broadly applicable cellular tool for studying temporal and spatial CDN signaling dynamics.
  • This biosensor facilitates research in infectious, malignant, and autoimmune diseases.