The efficiency of ethanol precipitation is strongly dependent on the length of the fragments smaller than 30�C40 nucleotides. The length dependence is a function of total salt and temperature and this can lead to low reproducibility with the small DNA fragments. These challenges can be overcome by the use of an enhanced hydroxyl protocol that was adapted from our prior work on duplex DNAs containing damaged sites and their complexes with drug like molecules. For the cleavage experiments the DNA was annealed in 100 mM NaCl, 10 mM KCl buffer to obtain the chair conformer. The drug like molecule was added to the annealed DNA sample. The cleavage reactions were then carried out and the DNA cleavage fragments purified. The cleavage in the presence of the drug like molecule was quantified and the results are displayed as percent change in cleavage in Figure 3. The cleavage results are also tabulated in File S4 for all of the concentrations examined. NSC 176319 appears to primarily alter the extent of cleavage of Y-27632 dihydrochloride residues 11, 12, 13 and 14 as depicted in Figure 3. The largest change is at residue 13 with more than 30% protection at the highest concentration examined. These four residues are NSC 136476 spatially close together in the chair structure. These results are consistent with NSC 176319 binding to a single site as discussed below. There is also some change in the cleavage at T9 that may be due to partial binding at a second site. The results indicate that NSC 91881 binding changes the extent of cleavage of the loop dT residues as well as some of the dG residues in the quartets. A single NSC 91881 is not nearly large enough to interact with all of these residues. A single NSC 91881 could interact with the dT residues since these two loops are spatially close together. Another binding site could be with the top loop. It appears that NSC 91881 binds to two or more sites of the 15 mer under these conditions as confirmed by the NMR results discussed below. The accessibility calculations predict more relative protection for the residues in the quartets than is observed in the cleavage reactions.