DNA-protein recognition has shown us the importance of DNA shapes in the recognition process. then reacted with 4M HCl/dioxane for 30 minutes to obtain the neomycin dimers as their hydrochloride salts in good yields (60%-71% yield for the two steps) (Figure 1). Ro 32-3555 4.2 FID assay reveals longer linker neomycin dimers are stronger binder than short linker dimers A single point FID assay23 was performed with the neomycin dimers and DNA duplexes ([5′-G2A6T6C2-3′] and [5′-A30T30-3′]). DNA duplexes were incubated with thiazole orange an exogenous intercalator at a stoichiometric ratio of 1 1: 0.5 (DNA base pair: thiazole orange) followed by the addition of neomycin dimers. The binding site size for neomycin dimer is ~12 base pairs/molecule therefore we used molar ratios of 1 1:1 and 1:5 for [5′-G2A6T6C2-3′] and [5′-A30T30-3′] respectively. The decrease in fluorescence intensity can be directly correlated with the affinity of neomycin dimers (Figure 2). In all the studies carried out including ITC and FID neomycin a Rabbit polyclonal to KIAA0174. weak binder of AT-rich DNA served as a control. There are few important trends that emerged from the FID study: (1) Neomycin dimers in general exhibit stronger binding to AT-rich DNA than neomycin (2) Neomycin dimers with long linkers specifically L= 16 to 22 exhibit 30-50 fold higher affinity to AT rich DNA sequences than neomycin. (3) Neomycin dimers (L=17-22) displayed higher affinity than short/rigid linker based neomycin dimers (L= 1 to 12). In the case of d[5′-G2A6T6C2-3′] DPA312 (L=19) attenuated the fluorescence intensity by a factor of ~25 than DPA71 (L=1). (4) The activity trend observed for neomycin dimers is similar towards both the AT-rich DNA target sequences however there is a slight difference in % fluorescence change which might be attributed to the difference in the DNA sequences. It appears from the FID assay that neomycin dimers DPA79 (L=18) and DPA312 (L=19) exhibit the greatest activity for the AT rich DNA duplexes. The significant decrease in the activity for neomycin dimers with short linkers (DPA71-DPA76) is presumably because of the decreased conformational freedom that the short linkers allow in comparison to the longer linkers. This would result in a decrease in the necessary flexibility required for optimal binding. Figure 2 Bar graph represents the FID-derived change in % fluorescence of AT-rich DNA-Thiazole Orange complex in the presence of indicated ligands. 4.2 The ITC-derived binding affinities of neomycin dimers corroborate with FID based binding activities ITC was employed to gain insight into the thermodynamics of binding between d[5′-G2A6T6C2-3′] and neomycin dimers (DPA71-DPA80) (Figure 3 ? 4 4 and supporting information Table S3). The ITC studies were performed at pH = 5.5 to ensure that the dimers were substantially protonated (lowest pKa for an amine group of neomycin = 5.91).19 The following observations were noted from the thermodynamic characterization. (1) Similar to our earlier reported work neomycin dimers with linker length L=12 to 22 exhibit two sets of binding sites in which the first is predominantly entropically driven with a binding stoichiometry of 1 1:1 (neomycin dimer:DNA) while the second is enthalpically driven with a binding stoichiometry of ~3:1 (neomycin dimer:DNA). The binding stoichiometry for the first binding event was ~10-12 base pairs per neomycin dimer which is also validated by CD and FID titrations in our earlier published work.13 (2) Ro 32-3555 Under otherwise matched conditions the ITC profile for neomycin dimers with linker length below L=12 is either not saturated or exhibits multiple Ro 32-3555 sets of binding Ro 32-3555 sites and precluded us from extracting any meaningful thermodynamic data (Fig. 4A). We conjecture this is due to weak binding interactions likely caused by electrostatic interaction between the multi-cationic neomycin dimers and the negatively charged DNA backbone. (3) DPA79 (L=18) with a binding constant Ro 32-3555 ~108 M?1 exhibits ~4-5 fold higher affinity than DPA72 (L=7). (4) ITC-derived binding constants for neomycin dimers with d[5′-G2A6T6C2-3′] agreed well with the FID assay based binding activity. Additionally the trend for ITC derived binding constant was in fair agreement with FID based AC50 values (aminoglycoside concentration required for 50% fluorescence change) calculated for neomycin dimer with.