Cell-Selective Multifunctional Surface Covalent Reconfiguration Using Aptamer-Enabled Proximity Catalytic Labeling

Abstract

Cell surface engineering provides access to custom-made cell interfaces with desirable properties and functions. However, cell-selective covalent labeling methods that can simultaneously install multiple molecules with different functions are scarce. Herein, we report an aptamer-enabled proximity catalytic covalent labeling platform for multifunctional surface reconfiguration of target cells in mixed cell populations. By conjugating peroxidase with cell-selective aptamers, the probes formed can selectively bind target cells and catalyze target-cell-localized covalent labeling in situ. The universal applicability of the platform to different phenol-modified functional molecules allows us to perform a variety of manipulations on target cells, including labeling, tracking, assembly regulation, and surface remodeling. In particular, the platform has the ability of multiplexed covalent labeling, which can be used to install two mutually orthogonal click reactive molecules simultaneously on the surface of target cells. We thus achieve “multitasking” in complex multicellular systems: programming and tracking specific cell–cell interactions. We further extend the functional molecules to carbohydrates and perform ultrafast neoglycosylation on target living cells. These newly introduced sugars on the cell membrane can be recognized and remodeled by a glycan-modifying enzyme, thus providing a method package for cell-selective engineering of the glycocalyx.

Multifunctional Proximity Labeling Strategy for Lipid Raft-Specific Sialic Acid Tracking and Engineering

“…(A) Schematic Representation of Multifunctional Proximity Labeling Strategy for Lipid Raft-Specific Protein and Sia Labeling a and (B) Extension and Application of the MLP Strategy b a HRP-CTxB is located specifically in the lipid rafts of cells, and HRP catalyzes DBCO-phenol (DP) labeling on lipid raft-specific proteins. 31 The C 7 site of Sia is then oxidized to an aldehyde group by NaIO4, and the oxidized aldehyde group undergoes an orthogonal reaction with hydrazidephenol (HP). The modified Sia is a precursor for HRP-mediated proximity labeling, while HRP catalyzes biotin-phenol (BP) labeling on lipid raftspecific Sia.…”

“…To address these issues, we sought to develop an extracellular covalent labeling method with spatial specificity. Proximity labeling strategies mediated by peroxidases, − biotin ligases, , and photocatalytics , have already been developed, which allow precise profile identification at different spatial specificities, such as cells, organelles, , neurosynapses, and protein microenvironments . Peroxidases, for example, horseradish peroxidase (HRP), , engineered ascorbate peroxidase (APEX), , and APEX2, mediate the conversion of phenol-containing molecules to highly reactive phenoxy radicals that covalently bind to electron-rich amino acids, such as tyrosine, in neighboring proteins. ,, The short lifetime (<1 ms) , and small labeling radius (<20 nm) of these radicals meet the necessary criteria for lipid raft-specific labeling.…”

“…Proximity labeling strategies mediated by peroxidases, − biotin ligases, , and photocatalytics , have already been developed, which allow precise profile identification at different spatial specificities, such as cells, organelles, , neurosynapses, and protein microenvironments . Peroxidases, for example, horseradish peroxidase (HRP), , engineered ascorbate peroxidase (APEX), , and APEX2, mediate the conversion of phenol-containing molecules to highly reactive phenoxy radicals that covalently bind to electron-rich amino acids, such as tyrosine, in neighboring proteins. ,, The short lifetime (<1 ms) , and small labeling radius (<20 nm) of these radicals meet the necessary criteria for lipid raft-specific labeling. The combination of proteomics and peroxidase-mediated proximity labeling provides a powerful tool for spatial proteomics. − From another perspective, in combination with fluorescent or functional molecules using bio-orthogonal reactions, peroxidase-mediated proximity labeling strategies lay the groundwork for localized tracking and engineering.…”

“…Peroxidases, for example, horseradish peroxidase (HRP), , engineered ascorbate peroxidase (APEX), , and APEX2, mediate the conversion of phenol-containing molecules to highly reactive phenoxy radicals that covalently bind to electron-rich amino acids, such as tyrosine, in neighboring proteins. ,, The short lifetime (<1 ms) , and small labeling radius (<20 nm) of these radicals meet the necessary criteria for lipid raft-specific labeling. The combination of proteomics and peroxidase-mediated proximity labeling provides a powerful tool for spatial proteomics. − From another perspective, in combination with fluorescent or functional molecules using bio-orthogonal reactions, peroxidase-mediated proximity labeling strategies lay the groundwork for localized tracking and engineering. Peroxidase is typically expressed at the desired location through gene transfection. − However, this process is time-consuming and labor-intensive, taking 24–48 h. To address this issue, receptor–ligand recognition methods offer a promising solution for the precise localization of peroxidases in a rapid and straightforward manner.…”

“…The combination of proteomics and peroxidase-mediated proximity labeling provides a powerful tool for spatial proteomics. − From another perspective, in combination with fluorescent or functional molecules using bio-orthogonal reactions, peroxidase-mediated proximity labeling strategies lay the groundwork for localized tracking and engineering. Peroxidase is typically expressed at the desired location through gene transfection. − However, this process is time-consuming and labor-intensive, taking 24–48 h. To address this issue, receptor–ligand recognition methods offer a promising solution for the precise localization of peroxidases in a rapid and straightforward manner. Cholera toxin B subunit (CTxB) has been used as the gold standard for identifying lipid rafts, as it specifically binds to GM1, a marker of lipid rafts. ,, Previous studies using FRET have detected GM1 and GPI-anchored proteins in these lipid rafts located at the submicron level in the plasma membrane …”