Conclusiones

Parallel Control Reactions Kept in the Dark. Figure 2 . S1 shows a series of negative control reactions that were not irradiated. In this experiment, 50 µ M concentrations of a bis-acridine 3 were pre-equilibrated for 1 h at 22 ºC with 38 µM bp of pUC19 plasmid DNA and 10 mM sodium phosphate buffer pH 7.0, in the absence and presence of one mol equiv of CuCl₂, 150 mM of NaCl, and/or 260 mM of KCl. The samples were kept in the dark for an additional 60 min at 22 °C and then electrophoreses on a 1.5

% non-denaturing agarose gel. (Fig.2.S1)

Figure 2 . S1. Photograph of a 1.5% nondenaturing agarose gel showing pUC19 plasmid DNA which was equilibrated in the presence and absence of bis-acridine 3 (no hν):

Lanes 1-3, no salt; Lanes 4-6, 150 mM NaCl; Lanes 7-9, 260 mM KCl; Lanes 10-12, 150 mM NaCl and 260 mM KCl. The samples contained 10 mM of sodium phosphate buffer pH 7.0, 38 µM bp DNA, and 0 µ M of 3 (Lanes 1, 4, 7, and 10) or 50 µM of 3 without (Lanes 2, 5, 8, and 11) and with (Lanes 3, 6, 9, and 12) 50 µM of CuCl2 (total volume 40 µL). The reactions were equilibrated for 1 h and 60 min in the dark (22 ºC).

2

Inhibition of DNA Photocleavage. In an attempt to identify possible reactive oxygen species (ROS) that contribute to DNA photocleavage, the following experiments were conducted using the singlet oxygen (¹O ) scavenger sodium azide and the hydroxyl radical (^•OH) scavenger sodium benzoate. Forty µL reactions containing

10 mM of sodium phosphate buffer pH 7.0, 38 µM bp pUC19 plasmid DNA, 10 µM of CuCl₂ and/or 10 µM of 3 or 50 µM of CuCl₂ and/or 50 µM of 4, in the presence and absence of either 100 mM of sodium azide or 100 mM of sodium benzoate were pre-

equilibrated in the dark for 1 h at 22 ºC. The samples were then irradiated for 60 min at 22 °C in an aerobically ventilated Rayonet Photochemical Reactor fitted with nine RPR-

4190 Å lamps. Reaction products were resolved on 1.5% non-denaturing agarose gels stained with ethidium bromide (0.5 µg/mL final concentration). The DNA on each gel was

visualized on a transilluminator set at 302 nm, photographed, and then quantitated using ImageQuant v. 5.2 software (Amersham Biosciences). For each lane on the gel, the percent totals of supercoiled, nicked, and linear plasmid DNAs within the lane were calculated. The percent inhibition of DNA photocleavage by 100 mM of sodium azide and by 100 mM of sodium benzoate could then be determined based on a comparison to the parallel reactions run in the absence of scavenger (Table S1). The following formulae were used.

Percent Inhibition of Nicked DNA = [(% Total of Nicked DNA without scavenger – % Total of Nicked DNA with scavenger)/(% Total of Nicked DNA without scavenger)] x 100

Percent Inhibition of Linear DNA = [(% Total of Linear DNA without scavenger – % Total of Linear DNA with scavenger)/(% Total of Linear DNA without scavenger)] x 100

Overall Percent of Photocleavage Inhibition = [(% Total of Linear and Nicked DNA without scavenger – % Total of Linear and Nicked DNA with scavenger)/(% Total of Linear and Nicked DNA without scavenger)] x 100

2

2

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Table 2 . S1. Percent inhibition of DNA photocleavage by the reactive oxygen species (ROS) scavengers sodium azide and sodium benzoate ^a

Scavenger ROS Compound Percent Percent Overall Percent of

Targeted Inhibition Inhibition Photocleavage of Nicked of Linear Inhibition

DNA DNA

a Individual photocleavage reactions consisting of 38 µM bp of pUC19 plasmid DNA in 10 mM sodium phosphate buffer pH 7.0 were pre-equilibrated with: (i) 10 µM of CuCl₂ and/or 10 µM of 3 or (ii) 50 µM of CuCl₂ and/or 50 µM of 4, in the presence and absence of 100 mM of sodium benzoate or 100 mM of sodium azide for 1 h at 22 ºC. The reactions were then irradiated at 419 nm for 60 min in an aerobically ventilated Rayonet

Photochemical Reactor.

b na = not applicable: no linear DNA was produced in the absence and presence of scavenger.

Thermal Melting Studies. Series of 1 mL solutions were prepared using 15 µM bp of calf thymus DNA (Invitrogen Cat. #15633-019) in 20 mM sodium phosphate buffer pH 7.0 or in 20 mM sodium phosphate buffer pH 7.0 with 10 µM or 2.5 of µM of

3 and/or 1 mol equiv CuCl₂ (Figs. S2, S3, S4). The solutions were placed in 1 mL (1 cm) quartz cuvettes (Starna) and were allowed to equilibrate in the dark for 1 h at 22 ºC.

The calf thymus DNA was then denatured by changing the temperature from 25 °C to 95 °C at a rate of 0.5 °C min^-1, while monitoring absorbance at 260 nm with a Cary-100 Bio UV-visible spectrophotometer (Varian). KaleidaGraph™ Version 4.0 software was used to calculate the first derivative of ∆A₂₆₀/∆T vs temperature, where the T_m value for

each melting isotherm was indicated by the maximum of the first derivative plot.

Figure 2.S2. Thermal melting curves and Tm values of 15 µ M bp calf thymus DNA in the absence and presence of 10 µM 3 and/or 10 µ M CuCl2 (20 mM sodium phosphate buffer pH 7.0). nd = not determined: no inflection point in isotherm and no maximum in

corresponding first derivative plot.

Figure 2.S3. Thermal melting curves and Tm values of 15 µM bp calf thymus DNA in the presence of 10 µM CuCl2 and/or 10 µM 4 or 10 µM 3 (20 mM sodium phosphate buffer pH 7.0). nd = not determined: no inflection point in isotherm and no maximum in corresponding first derivative plot. Compound 4 isotherms are from Fig.3, in Fernández et al., 2007.¹

Compound 3 isotherms are from Fig.2.S2, in this Supplementary data section.

Figure 2 . S4. Thermal melting curves and Tm values of 15 µM bp calf thymus DNA in the presence of 1 mol equiv of CuCl2 and/or 2.5 µM 3 (dashed lines) or 10 µM 3 (solid lines; 20 mM sodium phosphate buffer pH 7.0). nd = not determined: no inflection point in isotherm and no maximum in corresponding first derivative plot. The 10 µM Compound 3 isotherms are from Fig.2. S2, in this Supplementary data section.

CHAPTER 3.

PHYSIOLOGICALLY RELEVANT CONCENTRATIONS OF NaCl AND KCl INCREASE DNA PHOTOCLEAVAGE BY N-SUBSTITUTED