Ac dc

All nucleoside phosphoramidites including 5-methyl-dC, 5-hydroxymethyl-dC, 5-formyl-dC-III, 5-carboxy-dC, Ac-dC, dT, dA, dG, and dmf-dG, reagents, and controlled pore glass solid support for oligodeoxynucleotide synthesis were acquired from Glen Research Corporation (Sterling, VA). Human recombinant DNA methyltransferase 1 (DNMT1) and Δ580-DNMT1^{8 (link)} (missing the PCNA,^{23 (link)} DNMT3A/B interaction domains^{23 (link), 24 (link)}) were purchased from New England BioLabs (Ipswich, MA). Phosphodiesterase I, Phosphodiesterase II, and DNase I were acquired from Worthington Biochemical Corporation (Lakewood, NJ). Bovine intestinal alkaline phosphatase was procured from Sigma Aldrich Chemical Company (Milwaukee, WI). All remaining laboratory chemicals and solvents were purchased from ThermoFisher Scientific (Waltham, MA) and Sigma-Aldrich (Milwaukee, WI). The synthesis of ¹³C₁₀¹⁵N₂-5-Methyl-2′-deoxycytidine was described previously.^{22 (link)}

Standard phosphoramidites (Bz-dA, Ac-dC, dmf-dG, dT, and 5-Br-dU-CE) and controlled pore glass (CPG) having standard nucleosides were purchased from Glen Research (Sterling, VA). AEGIS phosphoramidites (dZ and dP) were obtained from Firebird Biomolecular Sciences LLC (Alachua, FL). All oligonucleotides containing Z and P (2P, 3/6ZP, 3/6ZP Br1 and 3/6ZP Br2, see Figure 1 and Table 1) were synthesized on an ABI 394 DNA Synthesizer following standard phosphoramidite chemistry, as previously reported.^{32 (link)} The CPGs carrying the synthetic oligonucleotides were treated with 1 M DBU in anhydrous acetonitrile (2.0 mL) at room temperature for 24 hours to remove the NPE group from the Z nucleobase. Then, the CPGs were filtered and dried. The CPGs carrying 2P and 3/6ZP (without 5-Br-dU) were treated with concentrated ammonium hydroxide at 55 °C for 16 hours, while the CPGs carrying 3/6ZP Br1 and 3/6ZP Br2 (with 5-Br-dU) were treated with concentrated ammonium hydroxide at room temperature for 24 hours. After removal of ammonium hydroxide, the oligonucleotides containing Z and P were purified on ion-exchange HPLC, and then desalted using Sep-Pac® Plus C18 cartridges (Waters). Fully standard oligonucleotides were purchased from Midland Certified Reagent Co. (Midland, Texas) in desalted form and used without further purification.