Conjugation of oligonucleotides to protein

Oligonucleotides were conjugated to ova by iEDDA-click chemistry (van Buggenum et al., 2016). Oligonucleotides were derivatized with trans-cyclooctene (TCO) in 10× borate buffered saline (BBS; 0.5 M borate, 1.5 M NaCl, pH 7.6; sterile filtered). Dilution of this buffer to 1× results in a final pH of 8.5. A reaction containing 40 nmol of amine-modified oligo (0.5 mM), 1× BBS, 10% DMSO, 8 µL of 100 mM TCO-PEG4-NHS in DMSO (10 mM final; Click Chemistry Tools, A137), pH 8.5 was rotated at room temperature for 15 min. A second aliquot containing the same amount of TCO-PEG4-NHS in DMSO was added, and the reaction was rotated at room temperature for another hour. Excess NHS was quenched by adding glycine, pH 8.5 to a final concentration of 20 mM and rotated at room temperature for 5 min. Modification was confirmed by analysis on an 8% denaturing TBE PAGE gel. Samples were precipitated by splitting the reaction into 20 µL aliquots and adding 280 µL of nuclease-free water, 30 µL of 3 M NaCl, and 990 µL of 100% ethanol. The precipitation reaction was incubated at −80°C overnight, followed by centrifugation at >10,000,000 ×g for 30 min. The supernatant was discarded, the pellet was washed with 100 µL of 75% ethanol, and centrifuged at >10,000,000 ×g for 10 min. The supernatant was removed, and the pellets were dried for 5 min at room temperature. The pellets were recombined by resuspension in 50 µL of 1× BBS. Samples were quantified by A₂₆₀.

To conjugate methyltetrazine to ova, detoxified ova (Sigma-Aldrich, St. Louis, MO) (using a Triton X-114 lipopolysaccharide detoxification method; Anis et al., 2007) was buffer exchanged into 1× BBS, pH 8.5. To an Amicon 0.5 mL 30 kDa filter (Millipore, UFC5030) was added 1 mg of ova and 1× BBS to a volume of 450 μL. The filter was centrifuged at 14,000 ×g for 5 min. The flow through was discarded and the sample washed twice with 400 µL of 1× BBS. The product-containing column was inverted into a clean collection tube and centrifuged at 1000 ×g for 2 min. Assuming no loss, the volume of the sample was adjusted to 2 mg/mL with 1× BBS. 400 µL of 1× BBS was added to the Amicon filter and stored at 4°C for later use. A 500 µL labeling reaction containing 0.5 mg of ova in 1× BBS and 50 µL of 2 mM mTz-PEG4-NHS in DMSO (0.2 mM final; Click Chemistry Tools, 1069), pH 8.5 was rotated at 4°C overnight. Excess NHS was quenched by adding glycine, pH 8.5 to a final concentration of 20 mM and rotated at room temperature for 10 min. The previously stored Amicon filter was centrifuged at 14,000 ×g for 5 min and the flow through discarded. 400 µL of reaction mixture was added to the filter and centrifuged at 14,000 ×g for 5 min. This was repeated until all 1 mg of protein had been added to the filter and was supplemented with 1× BBS as needed. Samples were washed 1× with 400 µL of 1× BBS. The product-containing column was inverted into a clean collection tube and centrifuged at 1000 ×g for 2 min. Assuming no loss, the volume of the sample was adjusted to 5 mg/mL with 1× BBS.

For the final antigen-DNA conjugation, a 100 µL reaction containing 300 µg of ova-mTz and 6 nmol of oligonucleotide-TCO (1:1 equivalents) in 1× BBS was rotated at 4°C overnight. Excess mTz was quenched with 10 µL of 10 mM TCO-PEG4-glycine and rotated at room temperature for 10 min. TCO-PEG4-glycine was prepared by reaction of 10 mM TCO-PEG4-NHS with 20 mM glycine, pH 8.5 in 1× BBS for 1 hr at room temperature and stored at −20°C. Products were analyzed by 10% TBE PAGE. For purification, excess ova and DNA were removed by filter centrifugation. 200 µL of 1× PBS was added to an Amicon 0.5 mL 50 kDa filter (Millipore, UFC5050) followed by 300 µL of sample. The filter was centrifuged at 14,000 ×g for 5 min and the flow through discarded. Samples were washed five times with 400 µL of 1× PBS and centrifuged at 14,000 ×g for 5 min. The product-containing column was inverted into a clean collection tube and centrifuged at 1000 ×g for 2 min. Purified products were analyzed by 10% TBE PAGE and total protein quantified with Bio-Rad protein quantification reagent (Bio-Rad, 5000006). LPS contamination after conjugation was below 0.5 EU/mg as mentioned in the 'Vaccinations' section.