Platinum 2

To obtain an activated Pt(DACH), 1 mM of dichloro(1,2-diaminocyclohexane) platinum(II) (Sigma-Aldrich, St. Louis, Mo, USA) was incubated with two molar equivalent of silver nitrate in the dark at 37°C for 18 hours. The resulting silver chloride was removed by centrifuging the reaction mixture at 13,000 g for 10 minutes after which the supernatant containing the activated oxaliplatin was recovered. For incorporation of aquated Pt(DACH) on DNA, each oligonucleotide with a single GpG site was mixed with 1.2-fold molar excess of activated Pt(DACH) in 10 mM sodium phosphate buffer (pH 6.8). The mixture was then incubated in the dark at 37°C for 18 hours. Purification of the Pt(DACH) containing oligonucleotide was done by an ion exchange column (Mono Q 5/50 GL, GE Healthcare) using a three-step gradient from 0.1 to 1.0 M NaCl in 10 mM Tris buffer, pH 8.0 (0–15% 1M NaCl buffer in 2 column volumes, 15–50% 1M NaCl buffer in 18 column volumes, and 50–100% 1M NaCl buffer in 0.5 column volume. The site-specifically platinated template DNA was desalted using Sep-Pak C18 cartridges (W aters) and dried with SpeedVac. The modified template was then reconstituted in water and annealed with the complementary upstream primer in 1:1 molar ratio by heating the mixture at 90°C for 10 minutes and slow-cooling over few hours at room temperature.

Platinum (II) (as K₂PtCl₄, purity ≥ 99.9%), 9,10-anthraquinone-2,6-disulfonic acid (AQDS, ≥ 98%) and rivoflavin (≥ 98%) were purchased from Sigma-Aldrich (St. Louis, MO, USA), and lawsone (2-hydroxy-1,4-naphthoquinone, > 98%) from TCI (Toshima, Kita-ku, Tokyo, Japan).