Cellular polynucleotides are alkylated by endogenous components, such as S-adenosylmethionine, or after reacting with two general classes of environmental and laboratory chemicals. SN1 chemical agents include alkylnitrosourea and N-alkyl-N-nitro-N-nitrosoguanidine that react with the N7 position of guanine, N3 of adenine, O6 of guanine, O2 or O4 of pyrimidines, and the non-phosphodiester oxygen atoms of the phosphate backbone. In contrast, SN2 chemical agents such as methyl methanesulfonate and dimethyl sulfate react primarily with the N1 position of adenine (1-Methyl-2'-deoxyadenosine) and N3 of cytosine. To avoid chain branching during synthesis when using DCI as activator, N6-Me-dA is offered with acetyl protection.
Coupling: No changes needed from standard method recommended by synthesizer manufacturer. Monomers that allow for UltraMILD deprotection are recommended (Catalog Numbers: dA: 10-1601-xx, dC: 10-1015-xx, dG: 10-1621-xx, dT: 10-1030-xx.) To avoid any exchange of the iPr-Pac group on the dG with acetyl, use the UltraMild Cap Mix A (40-4210-xx/ 40-4212-xx).
Deprotection: |If UltraMild Monomers were used: 0.05M Potassium Carbonate in Methanol, 4hrs at Room Temp.|DMT-Off Synthesis: Add 6uL acetic acid per mL 0.05M potassium carbonate in methanol. Dry under vacuum. Desalt per standard procedures. |DMT-On Synthesis: Use a Glen-Pak Purification Cartridge (60-5100-XX/60-5200-XX) to purify and desalt the oligonucleotide. Dilute the 0.05M potassium carbonate in methanol with 100mg/mL Sodium Chloride to a final concentration of 1:4(v/v). Load onto the prepped Glen-Pak Cartridge and purify as normal. |If Standard Monomers were used: |1) Prepare a solution of 10% DBU in anhydrous methanol. DBU (1,8-Diazabicyclo[5.4.0]undec-7-ene) is available from Aldrich (139009-25G) as is anhydrous methanol (322415-100ML). |2) Transfer the CPG to a clean, dry glass vial and add 1mL of the 10% DBU solution. |3) Seal the vial and leave for 5 days in the dark at room temperature. |4) Transfer the contents to a micro centrifuge tube and reduce under vacuum to a small volume. |5) Add 1mL of 10mM aqueous sodium hydroxide solution and desalt or purify the oligonucleotide using standard procedures.