Duplex DNA substrates used for the fluorescence-based experiments were prepared by annealing uracil-containing oligonucleotides with complementary strands containing 2AP opposite to the uracil. For the kinetic assays, a small excess of the 2AP-strand is preferable to an excess of the uracil-containing strand because the latter is also a substrate of UNG, and therefore its presence may affect the measured kinetic rates. For the kinetic assays, DNA substrates were prepared by annealing the strands at room temperature while monitoring the fluorescence intensity of 2AP in real-time. The uracil-containing strand was added to a known concentration of 2AP-containing strand, and the reduction in fluorescence intensity due to the formation of the duplex was measured until a small addition of the uracil strand did not result in a further decrease. The concentration of the resulting dsDNA substrate was calculated from the absorbance of the initial 2AP-containing strand and the volumes before and after adding the uracil strand. Traditional native polyacrylamide gel electrophoresis was used to confirm that the annealing procedure at room temperature was highly efficient and did not result in any measurable 2AP- or uracil-containing single strands. For the time-resolved and fluorescence quantum yield experiments, a slight excess of the U-strand is preferable to an excess of the 2AP-strand because 2AP in ssDNA is significantly brighter than 2AP in a duplex. Samples for these experiments were prepared as before and followed by the addition of a ~ 20% excess of the uracil-containing strand. In each case, we verified that further addition of the uracil-containing strand did not change the measured lifetimes or quantum yields. Duplexes for NMR experiments were prepared from complementary oligonucleotides mixed at 1:1 molar ratios that were heated to ~ 80 °C and annealed by cooling to room temperature for ~ 2 h.
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