FRAPPe - a novel FRET-based biosensor to detect inorganic polyphosphates

Abstract

Inorganic polyphosphate (polyP), a linear polymer of orthophosphate residues, is involved in a range of cellular processes, including phosphate storage, blood clotting, chromatin remodeling, RNA processing, and mitochondrial function. Despite its significance, tools for monitoring polyP with specificity and in real time are lacking, limiting insights into its dynamics. Here, we present FRAPPe (FRET-based Ratiometric Analysis of Polyphosphate), a FRET-based sensor for polyP. FRAPPe consists of an eCFP-eYFP FRET pair flanking CHAD, a polyP-binding domain. The sensor remains unresponsive to nucleic acids and other polyanions, while polyP binding induces a significant decrease in FRET, allowing quantitative estimation of polyP. We further show that FRAPPe can qualitatively estimate polyP levels from crude cell extracts, supporting its utility in high-throughput genetic and pharmacological screens to identify regulators of polyP metabolism and signaling.