Figure 2.

HPLC chromatograms (315 nm) of oligonucleotides digests (a) 11; (b) 12; (c) 13; (d) 14. Oligonucleotides were treated with DMC using optimized bifunctional activation conditions at 37°C. Enzymatic digestion: Oligonucleotides (1 A₂₆₀ unit) were incubated with 1 unit of nuclease P1 at 37°C for 2 hours in 0.8 mL of 0.02 M ammonium acetate, pH 5.5. The pH was adjusted to 8.2 by addition of 0.2 M NaOH. MgCl₂ (0.1 M solution, 20μL) was added followed by Snake Venom Diesterase (2 units) and Alkaline Phosphatase (2 units). Incubation continued at 37°C for 2.5 h. Digestion mixtures were analysed by HPLC on a Kromasil C-18 reverse phase column. The elution system was: 6-18% acetonitrile in 0.03 M potassium phosphate, pH 5.4, in 60 min, 1 mL/min flow rate. The temperature of the column was set at 45°C. More information is provided in the supporting information section (S-5-1 and S-4-2).