# FRET based imaging of cyclic dinucleotide dynamics in living systems

> **NIH NIH R21** · UNIVERSITY OF WASHINGTON · 2021 · $207,241

## Abstract

PROJECT SUMMARY
Cyclic dinucleotides of host and bacterial origin have emerged as ubiquitous second messengers
and potent modulators of host immune responses, with important roles in shaping infectious,
malignant and autoimmune diseases. The eukaryotic second messenger 2',3'-cGAMP is produced
by cGAS in response to DNA within the host cell cytoplasm. In response to DNA derived from
bacterial and viral infection cGAMP initiates host inflammation to clear infection, while sensing
of self-derived DNA has been implicated in autoimmune disorders including Systemic Lupus
Erythemytosus and Aicardi-Goutieres Syndrom. Additionally, bacteria produce a variety of cyclic
dinucleotides that function as second messengers and also promote host inflammation during
infection. In each of these instances, CDN binding to the mammalian receptor STING promotes
inflammatory responses. Despite our current understanding pertaining to CDN mediated
inflammation, there is a significant limitation in the capacity to directly measure and observe
CDNs within biological settings. To date, CDN detection relies on LC-MS/MS or ELISA based
methods. These technologies while important are limited in the spatial and temporal resolution
they afford. To overcome these current limitations, we have undertaken the development and
validation of a universal, genetically encoded fluorescent CDN biosensor. This sensor relies on
the CDN binding domain of STING and affords unparalleled temporal and single cell detection of
CDNs in living cells. We now aim to (i) biochemically characterize and establish the in vitro utility
of this sensor for monitoring CDN dynamics, (ii) utilize tissue culture studies to validate and
characterize the dynamics of CDN levels in living cells, and (iii) apply this new technology to
conduct a forward genetic screen for cell intrinsic regulators of cGAS-cGAMP signaling in human
cells. Together the studies outlined here will provide an innovative and broadly useful tool to
study CDN signaling within eukaryotes and provide potential biological insight into the regulation
of the cGAS-cGAMP signaling axis, with important consequences on infectious, malignant, and
autoimmune diseases.

## Key facts

- **NIH application ID:** 10183159
- **Project number:** 5R21AI153820-02
- **Recipient organization:** UNIVERSITY OF WASHINGTON
- **Principal Investigator:** Joshua Woodward
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $207,241
- **Award type:** 5
- **Project period:** 2020-07-01 → 2023-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10183159

## Citation

> US National Institutes of Health, RePORTER application 10183159, FRET based imaging of cyclic dinucleotide dynamics in living systems (5R21AI153820-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10183159. Licensed CC0.

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