(a) Schematic of ovalbumin (PDB code 1ova) antigen conjugation to barcoded DNA with phosphodiester and phosphorothioate DNA linkages and a 3′ biotin label (circle with B inside). Sulfur replaces a non-bridging oxygen to create a DNA phosphorothioate linkage. List of oligo sequences used can be found in Figure 1—source data 1. (b) Conjugation of oligonucleotides to ovalbumin. Purified conjugate was analyzed by 10% TBE native PAGE stained with GelRed for DNA (left) followed by Coomassie staining for protein (right). DNA-TCO: 61 nt barcoded oligonucleotide with 5′-trans-cyclooctene (TCO); ova-mTZ: ovalbumin functionalized with methyltetrazine (mTZ); ova-DNA: DNA-conjugated ovalbumin product with oligonucleotide attached. (c) Bone marrow-derived dendritic cells (BMDCs) were treated with pDNA, psDNA, ova-pDNA, or ova-psDNA (5 µg) by addition to the culture media. After 1, 3, and 7 days, cells were washed, released, lysed, and analyzed for pDNA or psDNA by qPCR. Values are displayed as fold-change relative to the negative control (cells alone). Asterisks denote sample significant amounts relative to the negative control (p<0.01; Wilcoxon rank-sum test). Error bars represent standard error of the mean (SEM). 3–5 wells were evaluated per group on 2–3 independent occasions. (d) Flow cytometric analysis of ova-psDNA conjugates acquired by BMDCs after 1 day or 7 days. Cells were washed 1 day after ova-psDNA treatment. Harvested BMDCs were stained with anti-ovalbumin made in rabbit and a secondary anti-rabbit conjugated to Phycoerythrin (PE) and then stained with streptavidin conjugated to brilliant violet 421 to visualize the 3′ biotin label on the psDNA. Shown are average and ± standard error. Experiment was performed three times with three technical replicates. (e) As in (d) except cells were plated onto glass coverslips and treated with ova-psDNA for 24 hr prior to staining with either anti-ovalbumin and a secondary conjugated to PE (red) followed by streptavidin conjugated to Fluorescein Isothiocyanate (FITC) (green). Co-localization is shown in yellow. Scale bar is 10 μm. Imaging was repeated three independent times. Approximately 100 cells were visualized with a similar frequency of double-positive cells as observed in (d). No single-positive cells were detected. (f) Analysis of DNAs as in (c) using murine lymph node lymphatic endothelial cells. (g) BMDCs were incubated with ova-psDNA (conjugated), ova plus psDNA (unconjugated), or PBS for 1, 3, and 7 days prior to adding OT-1 T cells labeled with violet proliferation dye. T cells and BMDCs were co-cultured at a ratio of 1:10 for 3 days. (h) Quantification of (g) using the percent divided calculation described in the Materials and methods. Experiments were performed three times with 3–5 wells per sample with similar results. Error bars represent SEM. Asterisks denote sample significant amounts relative to the negative control (p<0.05 Wilcoxon rank-sum test). Exact p-values are as follows: day 1 psDNA:ova-psDNA p=0.008, psDNA:untreated p=0.016, ova-psDNA:untreated p=0.016; day 3 psDNA:ova-psDNA p=0.008, psDNA:untreated p=0.016, ova-psDNA:untreated p=0.016; day 7 psDNA:ova-psDNA p=1, psDNA:untreated p=0.400, ova-psDNA:untreated p=0.400. n.d.: none detected.