Electronic Supplementary Material (ESM) to accompany: Perry CC, Urata SM, Lee M, Aguilera JA, Milligan JR. Radiation Protective Effects Produced by the Condensation of Plasmid DNA with Avidin and Biotinylated Gold Nanoparticles. Radiat Env Biophys.

Corresponding author contact info: email, ; phone: 858-534-4919; fax: 858-534-0265.

Figure S1. UV-vis spectra of pUC18 (10 g mL-1) and avidin (zero, 1, 3, 10, 30, and 100 g mL-1). The direction of increasing avidin concentration is indicated by the vertical arrow.

Figure S1 shows the UV-vis spectra of pUC18 and avidin. The UV-vis spectrum at the largest avidin concentrations shows the wavelength dependence characteristic of scattering by aggregates.

A / B
Figure S2. Representative topographic AFM images of pUC18 plasmid DNA in the absence (panel A) or presence of avidin (120 g mL-1, panel B). The horizontal scales in both images are 20 × 20 m. The vertical scales are 4 nm (panel A) or 25 nm (panel B).

Figure S2 reproduces representative topographic AFM images of pUC18 plasmid DNA in the absence or presence of avidin. In the absence of any avidin, the plasmid showed a typical filamentous appearance (Rackstraw et al. 2001). When avidin was present, numerous micron sized objects were clearly visible. However, since these AFM images were obtained with dried samples, the sizes of the particles may not be representative of those in aqueous solution.

Figure S3. Distribution of plasmid DNA and of avidin between pelletable particles and the remaining solution. The protein was either albumin (triangle) or avidin (all other data sets). After centrifugation, the fractions of plasmid DNA and of avidin present in the pellet (upper panel); the fraction of plasmid DNA present in the supernatant (middle panel); and the fraction of avidin present in the supernatant (lower panel) were all quantified. The plasmid concentration (closed symbol) was estimated either from its contribution to the UV absorption spectrum (circle), or by fluorescence of the cyanine dye BOBO-3 (cross, triangle). The avidin concentration (open symbol) was estimated from its contribution to the UV absorption spectrum (circle), from the change in the visible absorption spectrum of the azo dye HABA (square); or by the cuprous-bicinchoninate assay (rhombus).

Figure S3 shows the distribution of plasmid DNA and of avidin between pelletable particles and the remaining solution. The composition of the pellet was examined using UV spectroscopy after re-dissolving it in a solution containing 1 mol L-1 sodium chloride (in which it was soluble, see below). The concentrations of plasmid and avidin were estimated from the UV absorption between 250 and 290 nm. At avidin concentrations greater than 70 g mL-1, essentially all of the plasmid was present in the pellet. We note that the fraction of avidin in the pellet decreased from over 90% at 70 g mL-1 to ca. 30% at 300 g mL-1.

The composition of the supernatant remaining after centrifugation was assayed using several different methods. The plasmid was quantified by its UV absorption or by the fluorescence of the DNA binding cyanine dye BOBO-3 (Ruedas-Rama et al. 2010), which had been pre-bound to the plasmid prior to addition of avidin. At avidin concentrations less than 10 g mL-1, all of the plasmid was located in the supernatant. Increasing the avidin concentration from 10 to 50 g mL-1 gradually decreased the fraction of the plasmid to undetectably low (< 1%) levels. Further increases in the avidin concentration to 300 g mL-1 had no additional effect. If avidin was replaced by albumin, all of the plasmid remained in solution. The avidin concentration in the supernatant could be quantified by its UV absorption only if it was not masked by the larger contribution made by the plasmid. It was also detected by the change in the visible absorption spectrum after the post centrifugation addition of the azo dye HABA (Green 1965), and by a modification (Smith et al. 1985) of the Lowry assay (Lowry et al. 1951). The fraction of avidin remaining in the supernatant decreased to essentially zero at an initial avidin concentration of 60 to 70 g mL-1, consistent with its presence in the pellet. At higher initial avidin concentrations, an increasingly large fraction of it remained in solution, corresponding to the presence in the pellet of a constant amount of avidin equivalent to a concentration of 70 to 80 g mL-1, or a mass 7 to 8 fold greater than that of the plasmid.

Figure S4. Fraction of plasmid DNA remaining after centrifugation. The fraction of the plasmid still present in solution was estimated from the fluorescence intensity of the dye BOBO-3. The initial plasmid concentration was 10 (circle), 1 (square), or 0.1 (triangle) g mL-1. These measurements were made either in the presence (closed symbols, upper panel) or absence (open symbols, lower panel) of biotin. The biotin concentration was 1.5-fold greater than that of the binding sites present in avidin.

Figure S4 shows the effect of excess biotin on the fraction of plasmid DNA remaining after centrifugation. The plasmid concentration was assayed using the fluorescence intensity of pre-bound BOBO-3. The effect was examined at three different plasmid concentrations: 0.1, 1, and 10 g mL-1 (equivalent to nucleotide residue concentrations of 3.1×10-7, 3.1×10-6, and 3.1×10-5 mol L-1 respectively). To minimize any possible interference with avidin binding, the concentration of the DNA probe BOBO-3 was decreased to 3×10-9 mol L-1, at the cost of some decrease in the signal to noise ratio. At concentrations of 0.1, 1, and 10 g mL-1, the plasmid was completely pelleted by avidin concentrations of 0.6, 6, and 60 g mL-1(i.e., by a constant ratio of avidin to plasmid). There was no evidence that saturating the biotin binding sites had any effect on the ability of avidin to aggregate the plasmid into pelletable particles. This observation is not consistent with a report of a mild biotin dependent increase in the affinity of avidin for plasmid DNA, although at a higher ionic strength (Morpurgo et al. 2004).

Figure S5. UV-vis spectra of pUC18 (10 g mL-1), avidin (20 g mL-1), and Ac-R4-NH2 (zero, 1×10-6, 3×10-6, 1×10-5, and 3×10-5 mol L-1). The direction of increasing concentration Ac-R4-NH2 concentration is indicated by the vertical arrow.

Figure S5 shows the UV-vis spectra of pUC18, avidin, and Ac-R4-NH2. As in figure S1, the wavelength dependence at the higher ligand concentrations is characteristic of light scattering by aggregates.

1