Work

An Improved Ratiometric FRET Biosensor with Higher Affinity for Extracellular ATP

Year: 2025

Type: article

Abstract: Adenosine triphosphate (ATP) is readily released into the extracellular space as an autocrine and paracrine purinergic signaling molecule. We originally reported a genetically encoded, fluorescent protein-based Förster Resonance Energy Transfer (FRET) biosensor that can detect micromolar levels of extracellular ATP. Through mutagenesis of the ATP binding site and optimization of cell-surface display, here we report the development of a second-generation biosensor called ECATS2 with greater than three-fold higher affinity for extracellular ATP. We found that the tether length between the FRET biosensor and the cell surface anchor is critical to optimization of its performance. Furthermore, we demonstrate that the improved sensor can detect extracellular ATP release upon hypoosmotic stress in cultured astrocytes. This new sensor contributes an improved tool for the ratiometric detection of extracellular ATP dynamics and purinergic signaling.

Source: Sensors

DOI: 10.3390/s25185903

Authors: Autumn Cholger, Jason M. Conley, Elaine Colomb, Olivia de Cuba, Jacob Kress, Mathew Tantama

Institutions: Purdue University West Lafayette, Wellesley College

Language: English

Topic: Advanced Fluorescence Microscopy Techniques

Subfield: Biophysics

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $3,137

Funders: National Institutes of Health, National Institute of General Medical Sciences