Journal: bioRxiv

Article Title: ChemCell: Chemical Tethering of Large Biomolecules to Cell Surfaces through Diels-Alder Ligation

doi: 10.64898/2026.04.13.718024

Figure Lengend Snippet: Demonstration of the broad versatility of ChemCell technology. A) NK92-MI cells were treated with 1mM of Sia-2TCO for 48h, washed and subsequently reacted with a panel of Tz-decorated biomolecules of varying molecular weights. PEPTIDE) NK92-MI-TCO cells were incubated with 2.5 µM of Tz-FLAG or Tz-HA peptide for 30 min, washed and bound molecules were detected using anti FLAG or HA antibodies followed by staining with secondary fluorescent conjugates. Fluorescence was measured using flow cytometry. OLIGONUCLEOTIDE) NK92-MI were reacted with 2.5 µM of the hybridized DNA duplex containing two oligos with the tetrazine polyethylene glycol ( Tz-PEG ) moiety and Cy3 dye. In the latter experiment first modified cells with Tz-PEG containing single stranded oligonucleotide, followed by incubation with the complementary Cy3 containing ssDNA. As seen in the histogram of fluorescence, the cells could be successfully modified using both approaches. PROTEIN) NK92-MI cells were modified with TCO using the standard protocol. Then they reacted with recombinant protein G that was pre-decorated with PEG-Tz linker. The attached protein G was then visualized using mouse IgG-488 conjugate. IgG) NK92-MI cells were modified with TCO and then incubated with 4 µM Cetuximab modified with PEG-Tz linker. PROTEIN COMPLEX) R-phycoerythrin (PE), was first modified with Tz-PEG linkers using active ester chemistry ( Tz-PE ). Then 5 µM of Tz-PE was incubated for one hour with U2OS cells that were previously treated with 1mM of TCO for 48h. The attached protein has intrinsic fluorescence that was subsequently measured with flow cytometer upon detachment of cells from the dish. B) Benchmarking ChemCell to SPAAC using Tz-PE and DBCO-PE . U2OS cells were treated with 2mM SiaTCO, 2mM Sia-N 3 for 48hours, washed and reacted with 5 µM of either the Tz-PE or DBCO-PE. The reaction time was prolonged to 1 hour after which time, cells were detached from the dish, and the total cell fluorescence was measured by flow cytometry. C) Benchmarking ChemCell to SPAAC using Tz-Rituximab and DBCO-Rituximab . NK92-MI cells were modified with 1mM of both Sia-2TCO and Sia-N 3 for 48 hours, washed and reacted with different concentrations Tz-Rituximab or DBCO-Rituximab . The reaction time was prolonged to 1hour. Bound antibody was quantified with secondary anti human antibody and subsequently by flow cytometry. D) NK mediated cell lysis. NK92-MI cells were modified with 1mM Sia-2TCO and Sia-N 3 for 48 hours. Cells were washed and combined in 96 well plate with the target CD20 positive HG3 cells at 1:1 ratio. HG3 cells were labeled with fluorescein to distinguish them from NK92-MI cells. The target cell lysis was initiated by adding Rituximab (Tz or DBCO) in concentrations ranging from 0.008 µM up to 4 µM. After 3 hours of co-incubation cells stained with annexinV and the ratio of annexin positive target HG3 cells were measured using flow cytometry. We plotted the amounts of annexin negative cells as a function of added Rituximab. Marked decrease in viability was observed only when cells were labeled with Sia-2TCO and Tz-Rituximab was added to the mixture.

Article Snippet: 50 μL of R-phycoerythrin (precipitate 4 mg/mL solution in 60% saturated ammonium sulfate, 50 mM potassium phosphate, pH 7.0, ThermoFischer, P801) was pelleted at 25 000g/10 min.

Techniques: Incubation, Staining, Fluorescence, Flow Cytometry, Modification, Recombinant, Lysis, Labeling