| 2'-OMe-A-PACE Phosphoramidite |
PACE modifications have enjoyed a resurgence in interest as applied to the field of CRISPR gene editing. In an initial publication, it was shown that single guide RNAs (sgRNA) provided significantly higher activity in cells when 2'-O-methylthiophosphonoacetates were incorporated on the ends of the guide RNA to protect against cellular nucleases.1 In subsequent studies, 2'-OMe PACE modified sgRNAs were also shown to significantly increase on-target specificity of the CRISPR-Cas9 DNA cleavage in eukaryotic cells. In a recent paper, the incorporation of 2'-OMe PACE modified nucleotides in the 20-nucleotide guide region of the sgRNA was shown to decrease off-target cutting by over an order of magnitude while in most cases increasing the overall on-target efficiency as compared to unmodified single guide RNA.2
As an optimal cycle, we recommend using DCI as an activator (30-3150-XX) and a 15 minute coupling time. Following coupling, cap using Unicap with a regular coupling time and then oxidize using 0.5 M CSO for 3 minutes. Alternatively, a 33 minute coupling time using 0.45 M tetrazole, oxidation using low-water iodine (40-4032-XX) followed by capping with 6.5% DMAP as Cap B will give acceptable results. For deprotection, pre-treat the synthesis column with 1.5% DBU in anhydrous acetonitrile for 60 minutes at room temperature to remove 1,1-dimethyl-2-cyanoethyl protecting groups. Rinse the column with ACN, dry under argon and complete deprotection with 40% Methylamine for 2 hours at room temperature.
Formula: C46H58N5O10P
M.W.: 871.97
F.W.: 361.25
Diluent: Anhydrous Acetonitrile
Coupling: 15 minute coupling time using 0.25M DCI (30-3150-xx) is recommended. Using the phosphoramidite-based capping reagent Unicap (10-4410-xx), cap using a regular coupling time and then oxidize with 0.5M CSO for 3 minutes for best results.
Deprotection: Pretreat synthesis column with 1.5% DBU in anhydrous acetonitrile for 60 minutes at room temperature. Rinse with acetonitrile and dry with argon. Deprotect using 40% methylamine for 2 hours at room temperature.
Storage: Freezer storage, -10 to -30°C, dry
Stability in Solution: 2-3 days
DILUTION/COUPLING DATA
The table below shows pack size data and, for solutions, dilutions and approximate couplings based on normal priming procedures. Please link for more detailed usage information with the various synthesizers.
| ABI 392/394 |
| Cat.No. |
Pack
Size |
Grams/
Pack |
0.1M Dil.
(mL) |
LV40 |
LV200 |
40nm |
0.2µm |
1µm |
10µm |
| Approximate Number of Additions |
| Expedite |
| Cat.No. |
Pack
Size |
Grams/
Pack |
Dilution
(mL) |
Molarity |
50nm |
0.2µm |
1µm |
15µm |
|
| Approximate Number of Additions |
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