(15b) Light-Responsive Fluorescently-Labeled Cyclic Peptides for Selective and Reversible Labeling of Therapeutic Cells | AIChE

(15b) Light-Responsive Fluorescently-Labeled Cyclic Peptides for Selective and Reversible Labeling of Therapeutic Cells


Menegatti, S. - Presenter, North Carolina State University
Schneible, J., NC State University
Young, A., NC State University
Pozdin, V., NC State University
Freytes, D., NC State University
Daniele, M., NC State University UNC Chapel Hill
Abstract. One of the major challenges in mainstream cell-sorting technology is the removal of the affinity tags utilized for labeling the target cell population. Monoclonal antibodies targeting cell surface receptors, while highly selective, feature very high binding strength, which prevents their removal from the purified cells. To overcome this issue, we have developed a method for selective and reversible cell labeling, based on photo-switchable peptides that bind cell surface markers in an ON/OFF manner upon exposure to light. These peptides comprise a marker-targeting amino acid segment cyclized by an azobenzene linker, and tagged with a fluorescent dye. Exposure to light triggers cis/trans isomerization of the azobenzene linker, causing the peptide to switch reversibly between a binding and non-binding configuration. In this work, we have developed a fluorescein-labeled cyclic peptide specific for the surface marker VCAM1. Starting from a known VCAM1 binding linear peptide, an ensemble of azobenzene-cyclic variants were designed and optimized to ensure high binding selectivity and ON/OFF-type light-controlled binding. To this end, we combined in silico design using molecular dynamics and docking, with experimental sequence optimization by spectroscopy and surface plasmon resonance (SPR). The peptide lead cycloAZOB[G-VHAKQHRN-K] in particular displayed efficient photo-switching and ability to bind and release VCAM1 in solution with high yield upon exposure to light. Fluorescein-cycloAZOB[G-VHAKQHRN-K] was then utilized to label human umbilical vein endothelial cells (HUVECs), where VCAM1 expression was induced with glucose, in a light-dependent manner. The peptide, in fact, bound HUVECs in trans configuration, while showing negligible binding in cis, thereby demonstrating the potential of the proposed method for identifying peptides for cell labeling and purification.