Development of background-free tame fluorescent probes for intracellular live cell imaging.

Samira Husen Alamudi,Dongdong Su,Jihyo Kim,Christian Hoppmann,Haiyan Ren,Hee-Sung Park,Rajkumar Das,Hyung-Ho Ha,Lei Wang,Enrique Alvarado-Martínez,Lingna Hu,Young-Tae Chang,Jung Yeol Lee,Eduardo Peña-Cabrera,Rudrakanta Satapathy

doi:10.1038/ncomms11964

Abstract

Fluorescence labelling of an intracellular biomolecule in native living cells is a powerful strategy to achieve in-depth understanding of the biomolecule's roles and functions. Besides being nontoxic and specific, desirable labelling probes should be highly cell permeable without nonspecific interactions with other cellular components to warrant high signal-to-noise ratio. While it is critical, rational design for such probes is tricky. Here we report the first predictive model for cell permeable background-free probe development through optimized lipophilicity, water solubility and charged van der Waals surface area. The model was developed by utilizing high-throughput screening in combination with cheminformatics. We demonstrate its reliability by developing CO-1 and AzG-1, a cyclooctyne- and azide-containing BODIPY probe, respectively, which specifically label intracellular target organelles and engineered proteins with minimum background. The results provide an efficient strategy for development of background-free probes, referred to as ‘tame' probes, and novel tools for live cell intracellular imaging.

Highlights

Fluorescence labelling of an intracellular biomolecule in native living cells is a powerful strategy to achieve in-depth understanding of the biomolecule’s roles and functions
The model identifies three features: SlogP, logS and Q_VSA_FNEG, as key factors influencing cell permeability and nonspecific intracellular retention of small fluorescent probes. This new model is applied to design cyclooctyne- and azide-containing BODIPY ‘tame’ probes that can be utilized for labelling various intracellular organelles and engineered proteins via copper-free strain-promoted alkyne-azide cycloaddition (SPAAC)
To expedite an efficient strategy for ‘tame’ probe development, we set up a system to predict cellular properties of fluorescent probes via phenotypic high-throughput screening (HTS), cheminformatics and statistical analysis (Fig. 1a)

Summary

Introduction

Fluorescence labelling of an intracellular biomolecule in native living cells is a powerful strategy to achieve in-depth understanding of the biomolecule’s roles and functions. We report the first predictive model for cell permeable background-free probe development through optimized lipophilicity, water solubility and charged van der Waals surface area. The model identifies three features: SlogP (lipophilicity), logS (water solubility) and Q_VSA_FNEG (negatively charged van der Waals surface area), as key factors influencing cell permeability and nonspecific intracellular retention of small fluorescent probes. This new model is applied to design cyclooctyne- and azide-containing BODIPY ‘tame’ probes that can be utilized for labelling various intracellular organelles and engineered proteins via copper-free strain-promoted alkyne-azide cycloaddition (SPAAC)

Methods

Results

Conclusion