Oligodeoxynucleotides

Oligodeoxynucleotides were obtained from Integrated DNA Technologies, Inc. (IDT) (Coralville, Iowa) and Nuclease P1 (NP1) from Penicillium citrinum was from Sigma (St. Louis, MO). Ammonium citrate and 3-hydroxypicolinic acid (3-HPA) MALDI matrices were purchased from Fluka (Milwaukee, WI). HPLC solvents were from Fisher (Fair Lawn, NJ).

Peptides were custom synthesized by GenScript USA Inc. based on defined peptides with additional cationic arginine residues to increase molecular charge for electrostatic assembly. For myelin self-antigen, MOG, the MOG_35–55 peptide (MEVGWYRSPFSRVVHLYRNGK) was appended with 3 arginine residues and resulted in the sequence: MEVGWYRSPFSRVVHLYRNGKRRR. For the antigen control, ANT-CTRL, the GADp17 peptide (NMYAMLIARYKMFPEVKEKG) was appended with 9 arginine residues and resulted in a sequence: NMYAMLIARYKMFPEVKEKGRRRRRRRRR. GADp17 was selected for ANT-CTRL because it is a type 1 diabetes and has no known role in MS or the EAE model. Peptides were conjugated with fluorescein isothiocyanate (FITC) for loading calculations and were purified to >98% using HPLC. Oligodeoxynucleotides (ODNs) were custom synthesized by Integrated DNA Technologies, Inc. and include: TLR9 antagonist GpG (5-T*G*A*C*T*G*T*G*A*A*G*G*T*T*A*G*A*G*A*T*G*A-3) control ODN-CTRL (5-T*C*C*T*G*A*G*C*T*T*G*A*A*G*T-3), and TLR9 agonist CpG (5-T*C*C*A*T*G*A*C*G*T*T*C*C*T*G*A*C*G*T*T-3). ODNs were modified with a phosphorothioate bond modification to improve stability. Endotoxin analysis was performed for all components by the manufacturers. The charge approximations for the peptides and ODNs used in the study are shown in Fig. S3.

All primers and oligodeoxynucleotides used for the construction of shRNA plasmids were purchased from Integrated DNA Technologies. All shRNAs were cloned into the Age I/EcoR I sites of the pLKO.1 vector (Addgene, plasmid #10878) with a puromycin resistance cassette. All constructs were confirmed by Sanger sequencing. Scrambled shRNA with a hairpin sequence of 5′-CCT AAG GTT AAG TCG CCC TCG CTC TAG CGA GGG CGA CTT AAC CTT AGG-3′ (Addgene, Cambridge, MA, USA) was used as a negative control. Retrovirus was packaged in HEK293T cells by transfecting ALKBH4 shRNA-containing pLKO constructs together with two helper plasmids, that is, pCMV-dR8.2 and pCMV-VSV-G. Viral supernatants were harvested at 48 h following transfection and filtered before use. HEK293T and PC3 cells were subsequently infected with the harvested virus supernatants. After 24 h, the cells were treated for 72 h with fresh complete medium containing 1.0 and 0.4 μg/mL puromycin, respectively. The knockdown efficiencies were assessed by Western blot analysis.