Table 4.
Cleavage specificity of the ‘GCG’ design
| DNA cleavage site | EC50 (nM), I-MsoI ‘GCG’ | Predicted ΔEbinding |
|---|---|---|
| −8GCG/+6CGC (‘gcg’) | 29 | (0) |
| Alternative sites with palindromic single-base pair substitutions: | ||
| −8A/+8T | 206 | +2.4 |
| −8C/+8G | >1024 | +6.5 |
| −8T/+8A | >1024 | +2.8 |
| −7A/+7T | 310 | +8.6 |
| −7G/+7C | 68 | +6.7 |
| −7T/+7A | 24 | +4.5 |
| −6A/+6T | >1024 | +3.3 |
| −6C/+6G | >1024 | +2.4 |
| −6T/+6A | 507 | +1.8 |
Each indicated target site differs from the ‘gcg’ target site (Table 1) by corresponding single base pair changes on both sides of the palindromic target site. Top-stranded substitutions are indicated; complementary substitutions to the bottom strand are not shown. EC50 indicates the concentration of the endonuclease at which half of the target site was cleaved under the conditions described in ‘Materials and Methods’ section. The modeled binding energy is the predicted change in binding energy of the complex after repacking and minimizing the interface around each corresponding base pair substitution.