Adam M. Zahm, William S. Owens, Samuel R. Himes, Braden S. Fallon, Kathleen E. Rondem et al.
Cellular responses to stimuli underpin discoveries in drug development, synthetic biology, and general life sciences. We introduce a library comprising 6144 synthetic promoters, each shorter than 250 bp, designed as transcriptional readouts of cellular stimulus responses in massively parallel reporter assay format. This library facilitates precise detection and amplification of transcriptional activity from our promoters, enabling the systematic development of tunable reporters with dynamic ranges of 50−100 fold. Our library proved functional in numerous cell lines and responsive to a variety of stimuli, including metabolites, mitogens, toxins, and pharmaceutical agents, generating robust and scalable reporters effective in screening assays, biomarkers, and synthetic circuits attuned to endogenous cellular activities. Particularly valuable in therapeutic development, our library excels in capturing candidate reporters to signals mediated by drug targets, a feature we illustrate across nine diverse G-protein coupled receptors (GPCRs), critical targets in drug development. We detail how this tool isolates and defines discrete signaling pathways associated with specific GPCRs, elucidating their transcriptional signatures. With its ease of implementation, broad utility, publicly available data, and comprehensive documentation, our library will be beneficial in synthetic biology, cellular engineering, ligand exploration, and drug development.
Adam M. Zahm, William S. Owens, Samuel R. Himes, Braden S. Fallon, Kathleen E. Rondem, Alexa N. Gormick, Joshua S. Bloom, Sriram Kosuri, Henry Chan, Justin G. English. A massively parallel reporter assay library to screen short synthetic promoters in mammalian cells. 2024-11-28. https://doi.org/10.1038/s41467-024-54502-9
The synthetic promoter TRE-MPRA library was screened in HEK293 cells across additional metabolic, hormonal, heavy-metal, hypoxia-mimetic, and stress stimuli. Most treatment conditions were single biological replicates, with matched serum-free or vector-control baselines retained in the same table.
The barcoded TRE-MPRA library was transiently transfected into HEK293 cells for serum-free baseline, fetal bovine serum, and forskolin comparisons, and into five additional mammalian cell lines for serum-free versus fetal bovine serum benchmarking. The processed table retains promoter-level RNA/DNA activity for every available condition and cell-line context.
The TRE-MPRA library was used in HEK293 cells to profile transcriptional responses associated with nine GPCRs: ADRB2, DRD1, DRD2, HTR2A, OPRM1, PAR1, GPR91, MRGPRX2, and NTSR1. Receptor-expression controls, agonist treatments, endogenous ADRB2 stimulation, and NTSR1 signaling inhibition are represented as separate condition rows.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
Download all 12 files (ZIP)GSE271608_family.soft.gzGSE271608_finalBarcodeMap.csv.gzGSE271608_rpms.csv.gzREADME.txtsource_data.zipSupplementary_Data_1.xlsxSupplementary_Data_2.xlsxSupplementary_Data_3.xlsxSupplementary_Data_4.xlsxSupplementary_Data_5.xlsxSupplementary_Data_6.xlsxSupplementary_Data_7.xlsx