Issue 1, 2020

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

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

Article information

Article type
Paper
Submitted
19 صفر 1441
Accepted
28 ربيع الأول 1441
First published
29 ربيع الأول 1441

Mol. Syst. Des. Eng., 2020,5, 385-391

Author version available

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., 2020, 5, 385 DOI: 10.1039/C9ME00147F

To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements