SBIR Phase I: Time-Resolved Fluorescence (TRF) Microscopy of Live Cells with Cell-Penetrating Peptides and Other Targeting Agents

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This Small Business Innovative Research Phase I Project will link analogs of Lumi4®-Tb to various cell-penetrating peptides as a means of selectively delivering lanthanide probe molecules to living cells. The ability, in spatial and temporal dimensions, to dynamically image pharmaceutical and biologically relevant protein-protein, protein-DNA, and nucleic acid-nucleic acid interactions and kinetics in vivo using time-resolved microscopy is of significant value to pharmaceutical, diagnostic and drug-discovery industries. Using time-resolved microscopy (TRM), this project will analyze the CPP-probe conjugates for cell penetration and sub-cellular distribution. Lumi4-Tb is a new generation of luminescent lanthanide complexing agents that are exceptionally bright due to their high quantum yield (60%) and molar absorption coefficient (26,000 M-1cm-1). This proposal also will leverage unique TRM and protein labeling technology, based on the affinity of dihydrofolate reductase for trimethoprim. The combination of fluorescent lanthanide probes, FRET, and CPP-mediated cell delivery will make it possible to dynamically detect and stoichiometrically quantify protein-protein interactions in live cells with unprecedented sensitivity.The broader/commercial impact of this project, if successful, will be to develop a new class of cell imaging reagents and techniques that will improve the ability of researchers to follow protein-protein interaction pathways with quantitative accuracy that has not been available before. This will impact not only fundamental and applied research but also primary healthcare through the discovery of novel pharmaceutical targets and mechanisms to diagnose and treat disease. The design and use of novel probes to study structure and function at the molecular and subcellular level in living cells is a topic of great importance, with a growing need for new approaches and tools to visualize not only the distribution of molecular species in cells, but the manner in which they interact. Protein-protein interactions and other dynamic events within cells have been largely invisible, but will be increasingly observable with new imaging modalities. In particular, lanthanide probes, with the dramatic lowering of background achieved through time gating can enable new microscopic imaging if coupled successfully with cell penetration and molecular targeting and recognition.