Toward Cell‐Permeable, Multi‐Fluorophore Protein Labels for Enhanced LRET Imaging.

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 Tb(III) 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 ns‐scale fluorescence background and cleanly detect Tb(III)‐sensitized FP emission. In order to increase LRET signals and achieve faster and more sensitive imaging, we have sought to develop biosensors that combine multiple Tb(III) donors with a single fluorescent acceptor. Due to the mismatch in lifetimes, multiple excited donors can successively transfer energy to one acceptor, thereby generating many photons with a single excitation. Our lab has previously shown that heterodimers of a protein‐targeting ligand linked to a Tb(III) complex can be efficiently delivered to the cytoplasm of living cells by conjugating the heterodimers to arginine‐rich, cell penetrating peptides (CPPs) such as nonaarginine or Tat. Our results are in line with numerous studies which show that CPPs facilitate direct translocation of small molecules from culture medium into the cytoplasm. Macromolecules linked to CPPs enter cells via endocytosis, and the maximum size of CPP cargo that can bypass endocytosis and directly enter cytoplasm is unknown. In order to resolve this question and inform our design of multi‐Tb(III) probes, we conjugated oligoarginine to polyamidoamine (PAMAM) dendrons of different generations. We used FMOC‐based, solid‐phase peptide synthesis (SPPS) to prepare nonaarginine that was N‐terminally coupled to PAMAM dendrons with 2, 4, and 8 branches (generations 0, 1 and 2, respectively). Each branch terminates in a primary amine, which was coupled to 5,6‐carboxy fluorescein. Fluorescence microscopy showed that 2‐dye and 4‐dye Dendron conjugates entered the cytoplasm of HeLa cells within 30 min when cells were incubated at 4 °C or 37 °C in culture medium containing 5 μM or higher probe concentrations. The 8‐dye Dendron‐CPP conjugate did not enter the cytoplasm under any set of incubation conditions. Based on these results and published values for PAMAM dendrimer sizes, we estimate the molecular (hydrodynamic) diameter cut‐off for CPP‐mediated cytoplasmic delivery to be < 2 nm. Support or Funding Information Funding provided by the National Institutes of Health, R01 GM081030.