Table 1. Equilibrium and kinetic constants for the interaction of compounds 1 and 3 with different sequence DNAs by SPR analysisa.
Keqb (×10–7) | kac (×10–5) | kdc (×103) | ka / kd (×10–7) | |
---|---|---|---|---|
1-AATT | 9.4 | 0.61 | 1.7 | 3.6 |
1-AT | 6.3 | 1.1 | 1.3 | 8.1 |
1-GC | <1 | |||
3-AATT | 2.1 | 3.0 | 12 | 2.6 |
3-AT | 2.0 | 3.4 | 5.1 | 6.6 |
3-GC | 0.18 | 0.47 | 25 | 0.20 |
We estimate the errors in BIA equilibrium constants for the single binding sites in this study to be: (i) for K values between ∼1 × 105 and ∼2–3 × 107, which is the most accurate range, errors are <10%; (ii) for K values between ∼5 × 103 and ∼5 × 104 errors increase to ∼15–20% due to the difficulty of reaching RU values close to the site saturation point; (iii) for K values >2–3 × 107 errors again increase, due to the difficulty of collecting data at low amounts of site saturation. We estimate the error in the AT binding constants for compound 1 to be ∼10–15% and for compound 3 to be ∼10%. The error in the K for GC binding of compound 3 is ∼10–15%. These error estimates are in agreement with K values from repeat BIAcore experiments.
aExperiments were conducted in HBS buffer at 25°C.
bEquilibrium constants were determined from the RU values in the steady-state region of the sensorgrams at each concentration as described in the Materials and Methods.
cKinetic constants were determined by global fitting of sensorgrams at all concentrations in each experiment.