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Design of protein-based “turn on” molecular probes for intracellular bond cleavage

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Abstract

The clinical success of antibody-drug conjugates and numerous other stimuli-responsive drug delivery systems motivates the need to develop molecular tools to quantitatively study their intracellular processing. To this end, researchers have developed fluorescent-based probes utilizing Förster resonance energy transfer (FRET). Quenched probes have the potential to eliminate fluorescence bleed through, which is common in FRET-based systems. However, the hydrophobicity of many broad-spectrum fluorescence quenchers can complicate the design of protein-based molecular probes. In this work, we investigate the potential for 2,4-dinitroaniline (2,4-DNA) to serve as a hydrophilic fluorescence quencher. A support-free synthesis of oligothioetheramide (oligoTEA) linkers was developed and applied to the design of quenched fluorescence probes. These quenched probes were based on intramolecular static quenching of boron dipyrromethene (BODIPY)-FL by 2,4-DNA. Probes containing a reduction-sensitive disulfide bond and a protease-sensitive valine–citrulline-PABC linker were synthesized using a model protein – human transferrin. Within HeLa cells, the apparent degradation rate of the disulfide bond was greater than the valine–citrulline-PABC linker. This work establishes a versatile method for synthesizing multifunctional crosslinkers and identifies 2,4-DNA as an effective fluorescence quencher for protein-based bioconjuates.

Graphical abstract: Design of protein-based “turn on” molecular probes for intracellular bond cleavage

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Publication details

The article was received on 18 Oct 2019, accepted on 25 Nov 2019 and first published on 26 Nov 2019


Article type: Paper
DOI: 10.1039/C9ME00147F
Mol. Syst. Des. Eng., 2019, Advance Article

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    Design of protein-based “turn on” molecular probes for intracellular bond cleavage

    M. R. Sorkin, J. A. Walker, F. Ledesma, N. P. Torosian and C. A. Alabi, Mol. Syst. Des. Eng., 2019, Advance Article , DOI: 10.1039/C9ME00147F

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