Antibody conjugation with DNA oligos

Secondary antibodies were conjugated to docking strand (DS; see Supplementary Table ¹ for the sequences) oligos via DBCO-azide click chemistry. First, DS oligos were conjugated to either DBCO-PEG12-NHS ester, DBCO-PEG4-NHS ester, or DBCO-Sulfo-NHS ester (no PEG). DBCO-ester was added in 20x molar excess to the DNA in a total reaction volume of 50 uL. The reaction ran for 3 h at room temperature and was carried out in ultra-pure water, pH adjusted to 8.5 with 1 M sodium bicarbonate. After the reaction, ethanol precipitation with 0.3 M sodium acetate at −80 °C was repeated twice on the mixture to purify the DS-DBCO product. Final DS-DBCO products were suspended in ultra-pure water. To prepare antibody-PEG4-azide conjugates, azido-PEG4-NHS was added in 100x molar excess to secondary antibodies; the reaction was carried at pH~8.5 adjusted by 1 M sodium bicarbonate and ran for 3 h at room temperature. Antibody-PEG4-azide conjugates were flowed through a 50 kDa size exclusion column and washed with PBS 15 times on the column via centrifugation 4 °C (6000 g, 2.5 min each).

Next, DS-DBCO was reacted in 5x molar excess to the antibody-PEG4-azide via copper-free click chemistry; the reaction took place overnight on a shaker at room temperature. The resulting antibody-PEGx-DS (x = 4, 8, or 16 depending on the PEG linker of DS-DBCO) product was purified by flowing through a 100 kDa size exclusion column and washing in PBS 5 times by centrifugation (at 6000 g, 2.5 min each, 4 °C). The final product (antibody-PEGx-DS) was suspended in PBS. Product concentrations were measured with a NanoDrop UV-Vis spectrophotometer (ThermoFisher Scientific, 2000c). Peak signals at 280 nm, 495 nm (for 6-FAM), or 550 nm (for Cy3^™) were used to calculate the protein concentrations and the degrees of labeling. The antibody-PEGx-DS used in this work typically had a degree of labeling of 4–5 (i.e., 4–5 DS oligos per antibody).