LRET Imaging Improvement with Cell-permeable, Multi-fluorophore Protein Labels

Genetically encoded biosensors based on Förster Resonance Energy Transfer (FRET) are used to microscopically visualize dynamic changes in protein interactions and activities within living cells. However, the spectral overlap between fluorescent protein (FP) donors and acceptors makes it challenging to accurately quantify changes in FRET signals, and multi-color imaging of two or more FP FRET pairs in a single cell is extremely difficult. Lanthanide-based FRET (LRET) biosensors incorporate luminescent Tb3+ or Eu3+ complexes with ms-scale excited-state lifetimes and multiple narrow-line emission bands as donors and FPs (or other conventional fluorophores) as acceptors. Time-gated microscopy uses pulsed excitation and delayed detection to eliminate the ns-scale fluorescence background and cleanly detect Ln(III)-sensitized FP emission. To increase LRET signals and achieve faster and more sensitive imaging, I have sought to develop biosensors that combine multiple Ln(III) donors with a single fluorescent acceptor. Due to a mismatch in emission lifetimes, multiple excited donors can sequentially transfer energy to a single nearby acceptor, thereby generating several photons following a single excitation pulse. Numerous studies have demonstrated that cell-penetrating peptides (CPPs) facilitate direct translocation of small molecules from culture medium into the cytoplasm. This dissertation includes the results of efforts to design and prepare small molecule protein labels that are linked to multiple lanthanide complexes and CPPs as part of a strategy to improve the cellular uptake of bright LRET biosensors. Although the mechanism of cellular entry is under debate, there is clear evidence that shows that small molecules linked to CPPs can translocate directly into the cytoplasm from culture medium in a non-endocytic manner while macromolecules enter cells via endocytosis. The extent to which CPP-linked cargo enters via an endocytic or non-endocytic pathway depends on several factors including cargo size, temperature, concentration, and plasma membrane lipid composition. In this dissertation, I present the results of efforts to design and characterize lanthanide probes that are comprised of multiple Tb3+ or Eu3+ complexes that are conjugated to CPPs. I show that a probe bearing two Tb3+ complexes effectively doubled the sensitized emission intensity relative to equivalent single-luminenophore probes. By adjusting cell-loading temperature and culture medium composition, I successfully delivered dual-Tb3+ probes into the cytoplasm and selectively labeled recombinantly expressed fusion proteins, thereby demonstrating proof-of-concept of my overall LRET signal-enhancement strategy. In the final chapter, I present new LRET probe designs including a Tb3+ complex linked to tris-nitrilotriacetate for labeling histidine-tagged proteins and a Eu3+ protein label.