(640e) A Genetically Encoded Fluorescent Reporter for Polyamines

Polyamines are essential metabolites found across all kingdoms of life. They are involved in a variety of cellular processes, including growth, differentiation, immune function, aging, and cell death. Dysregulation of polyamine metabolism is linked to several pathological conditions, such as cancer, neurodegeneration, diabetes, and inflammation, but the precise biochemical events underlying these associations remain unclear. To better understand polyamine metabolism and to develop effective interventions for these diseases, it is necessary to develop methods for the sensitive and specific measurement of polyamines in biological systems. Current methods rely on chromatography or mass spectrometry (MS)-based detection. However, these methods have several crucial limitations. First, these methods require large quantities of samples (~million cells) and have limited throughput. Cell-to-cell heterogeneity is lost, and the data report on average polyamine content over the sample. Two, these methods necessitate polyamine extraction from their native cellular context using organic solvents and extensive chemical derivatization. Thus, the measurements are confounded by polyamine loss during extraction and labeling efficiencies. The commonly used polyamine adducts are also light-sensitive, rendering them susceptible to degradation during injection and separation. Finally, polyamines are predominantly bound to nucleic acids and exchange slowly (over days), which means that even significant changes in biologically active polyamine pools may be missed as experimental noise in total polyamine determinations. To address these issues, we have developed a genetically encoded fluorescent polyamine reporter by re-engineering the cellular polyamine detection machinery. This biosensor allows quantitative measurement of polyamine levels at single-cell resolution and has allowed us to study polyamine metabolism in heterogenous tissues and conduct large scale genetic screens. This reporter has been successfully used across a variety of human cell lines and tissues. Additionally, we observed an average inter-day assay coefficient of variations (CVs) of only ~3% compared to the reported gold standard for LC/MS (~20-30% ). The simplicity of the assay significantly improves laboratory throughput to characterize polyamine in hundreds of thousands of samples. From our experience, one individual can analyze four plates of 384 live samples (in total, ~1500 unique perturbations) per hour by hand with multi-channel pipettes and a benchtop automated microscope. This is significantly faster than LC/MS which would entail a 6-7 days protocol and can be further fully automated using a robotics platform.