Phosphonoacetate (PACE) modified oligonucleotides show great potential as biological modifiers in a wide variety of research applications. PACE monomers are part of a family of Phosphonocarboxylate monomers. The monomers can be easily incorporated into complex oligonucleotides and are compatible with a wide variety of other sugar or heterobase modifications. PACE DNA can be conjugated through the carboxylic acid functional group. They have been shown to be active in siRNA duplexes and accelerate the initial rate of cleavage by RNase H-1 when incorporated with phosphorothioates. However, the most interesting observation to date is that they exhibit an unprecedented enhancement in penetration of cultured cells.
The phosphonoacetates are fully soluble in acetonitrile at a recommended concentration of 0.1M and are compatible with standard DNA synthesizers. A recommended coupling time of 33.3 minutes with 1H-Tetrazole is necessary when using the standard protocol. A modified LV cycle for AB instruments that reduces coupling time to 15 minutes with 1H-Tetrazole is available on our website. Oxidation must precede capping in the synthesis cycle. Reagents for oxidation depend on the type of synthesis. For fully modified oligos, we recommend the non-aqueous oxidizer camphorsulfonyloxaziridine (CSO) as a 0.1M solution. For mixed phosphodiester and phosphonoacetate modified oligos, a 0.5M CSO solution is recommended. Low water oxidizer, 40-4032, is an alternative oxidizing reagent although it has been reported that this can result in conversion of a small percentage of the phosphonoacetate to the phosphodiester. We also recommend the use of the Cap Mix B with DMAP (40-4020) instead of the standard Cap Mix B containing 1-Methylimidazole.
The standard protocol for cleavage and deprotection requires a two step method with pretreatment using 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) and subsequent cleavage using methylamine. The DBU is used to deprotect the dimethylcyanoethyl (DMCE) protecting groups and to prevent alkylation of the bases during deprotection. Cleavage with 40% methylamine in water is recommended and we have also had good results when using AMA deprotection.
Phosphonoacetate (PACE) modified oligonucleotides show great potential as biological modifiers in a wide variety of research applications.
PACE modifications have enjoyed a resurgence in interest as applied to the field of CRISPR gene editing.