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Abstract Photo‐affinity adsorbents (i.e., translucent matrices functionalized with ligands featuring light‐controlled biorecognition) represent a futuristic technology for purifying labile biologics. In this study, a framework for prototyping photo‐affinity adsorbents comprising azobenzene‐cyclized peptides (ACPs) conjugated to translucent porous beads (ChemMatrix) is presented. This approach combines computational and experimental tools for designing ACPs and investigating their light‐controlled isomerization kinetics and protein biorecognition. First, a modular design for tailoring ACP's conformation, facilitating sequencing, and streamlining the in silico modeling of cis/trans isomers and their differential protein binding is introduced. Then, a spectroscopic system for measuring the photo‐isomerization kinetics of ACPs on ChemMatrix beads is reported; using this device, it is demonstrated that the isomerization at different light intensities is correlated to the cyclization geometry, specifically the energy difference of trans versus cis isomers as calculated in silico. Also, a microfluidic device for sorting ACP‐ChemMatrix beads to select and validate photo‐affinity ligands using Vascular Cell Adhesion Molecule 1 (VCAM‐1) as target protein and cycloAZOB[GVHAKQHRN‐K*]‐G‐ChemMatrix as model photo‐affinity adsorbent is presented. The proposed ACPs exhibit rapid and defined light‐controlled isomerization and biorecognition. Controlling the adsorption and release of VCAM‐1 using light demonstrates the potential of photo‐affinity adsorbents for targets whose biochemical liability poses challenges to its purification.
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Photoinduced reconfiguration to control the protein-binding affinity of azobenzene-cyclized peptides
The impact of next-generation biorecognition elements (ligands) will be determined by the ability to remotely control their binding activity for a target biomolecule in complex environments. Compared to conventional mechanisms for regulating binding affinity (pH, ionic strength, or chaotropic agents), light provides higher accuracy and rapidity, and is particularly suited for labile targets. In this study, we demonstrate a general method to develop azobenzene-cyclized peptide ligands with light-controlled affinity for target proteins. Light triggers a cis/trans isomerization of the azobenzene, which results in a major structural rearrangement of the cyclic peptide from a non-binding to a binding configuration. Critical to this goal are the abiliy to achieve efficient photo-isomerization under low light dosage and the temporal stability of both cis and trans isomers. We demonstrated our method by designing photo-switchable peptides targeting vascular cell adhesion marker 1 (VCAM1), a cell marker implicated in stem cell function. Starting from a known VCAM1-binding linear peptide, an ensemble of azobenzene-cyclized variants with selective light-controlled binding were identified by combining in silico design with experimental characterization via spectroscopy and surface plasmon resonance. Variant cycloAZOB[G-VHAKQHRN-K] featured rapid, light-controlled binding of VCAM1 (KD,Trans/KD,Cis ~ 130). Biotin-cycloAZOB[G-VHAKQHRN-K] was utilized to label brain microvascular endothelial cells (BMECs), showing co-localization with anti-VCAM1 antibodies in cis configuration and negligible binding in trans configuration.
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- PAR ID:
- 10168676
- Date Published:
- Journal Name:
- Journal of Materials Chemistry B
- ISSN:
- 2050-750X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/D0TB01189D
