Radiation Target Analyses of Plasmid DNA - American Chemical Society

Chapter 17

Radiation Target Analyses of Plasmid DNA 1

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Downloaded by UNIV OF CALIFORNIA SAN DIEGO on March 12, 2017 | http://pubs.acs.org Publication Date: December 21, 2007 | doi: 10.1021/bk-2007-0978.ch017

T. J . Anchordoquy , Μ· D. C. Molina , and E . S. Kempner

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School of Pharmacy, University of Colorado Health Sciences Center, Denver, CO 80262 Office of Science and Technology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892 2

Frozen solutions of high molecular weight circular DNA were irradiated with high-energy electrons. Molecules that survived the radiation exposure were detected by structural and functional techniques and were analyzed using radiation target theory. Radiation damage (as a loss of function) was not observed in a distant portion in the same chain, indicating a lack of energy transfer along the DNA polynucleotide. The results complement the same phenomena observed in RNA but are in contrast to the observations in proteins.

Effects of ionizing radiation on DNA have been widely studied for many years (1), although previous radiation target analyses were only reported before well-defined preparations and advanced techniques became available. It was reported that radiation damage in RNA occurred only locally in that molecule (2), in distinction with the well-established spread of radiation damage throughout polypeptides (3). Gamma rays and high-energy electrons ionize atoms at random along their trajectories; the chance of an interaction being dependent on the number of atoms in the molecule. In each interaction, large amounts of energy are transferred to the target, resulting in excitations and covalent bond breakage. These direct effects of radiation dominate infrozenor dried materials. Because of the randomness, the undamaged (surviving) molecules decrease exponentially

U.S. government work. Published 2008 American Chemical Society.

Celina and Assink; Polymer Durability and Radiation Effects ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

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196 with radiation exposure; the rate of this exponential is directly proportional to the mass of the target molecules. A circular form of DNA (plasmid) encoding the gene for green fluorescent protein (GFP) was used in the present experiments. The DNA structure in plasmids is the classical double-helix form in which two strands of DNA interact via hydrogen bonds and base-stacking interactions. The GFP plasmid contains 5030 base pairs (bp) of total mass 3300 kDa. When produced in bacteria, the circular double-stranded plasmid is highly coiled into a compact "supercoiled" form (Figure 1). A single break in the backbone of either of the two DNA

Figure 1. The two strands of the classical DNA double helix (left) are circularized (center) and twisted into a supercoiled form (right) when produced in bacteria.

strands causes the double-stranded molecule to unwind its supercoils and assume a relaxed, open-circular form. If a second break occurs in the other DNA strand nearby the location of the first break, the resulting double-strand break will cause the circular form to be converted to a linear form. These three forms have very different hydrodynamic properties and can readily be separated by agarose gel electrophoresis. In addition to a physical separation and quantification of the different plasmid forms, the biological activity of the plasmid can be assessed by introducing irradiated DNA into cells and monitoring the levels of GFP expression. These results revealed that in a single direct radiation interaction, damage is confined to a limited region of the DNA molecule.


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Several earlier radiation studies of plasmid DNA have appeared, but only two concerned the direct actions of ionizing radiation; both used very low energy electrons. Hieda et al. (4) studied radiation interactions directly with the phosphorus atom; in spite of the large number of orbital electrons, phosphorus ionizations were not unusually effective in radiation damage. Boudaiffa et al. (5) showed that electrons with energies typical of secondary electrons could cause single-, double- and multiple-strand breaks in DNA.

Materials and Methods The plasmid encoding green fluorescent protein (pGreen Lantern-1) was originally obtained from Gibco-BRL (Carlsbad CA). The plasmid was propagated in E. coli, and purified by Aldevron Custom Plasmid Purification (Fargo ND). The plasmid was dissolved at a concentration of 3 mg/mL in sterile 2.5 mM Tris-HCl pH 8.5. Plasmid samples (20 uL) were frozen on dry ice in 2 mL glass ampules and sealed with an oxygen-gas flame. Sealed, frozen samples were shipped and held at -80 C before and after irradiation at -135 C (6) with 13 MeV electrons from a linear accelerator at the Armed Forces Radiobiology Research Institute (Bethesda MD) or at the National Institutes of Standards and Technology (Gaithersburg MD).

Assays: Conversion of supercoiled plasmids to the open circle and linear forms were monitored by subjecting samples to 0.9% agarose gel electrophoresis (7). Unirradiated plasmid ranging from 100 to 1000 ng was included on each gel with irradiated samples in order to create a standard curve. After migration, gels were stained with ethidium bromide and quantified by fluorometric image analysis using a Fluor-S Multilmager and Quantity One software (Biorad, Hercules CA). Aliquots of irradiated plasmid containing 2 ug DNA were diluted in water and mixed with 8.4 uL of 10 mM polyethylenimine (pH 7) to facilitate delivery to African green monkey kidney cells (COS-7) as previously described (8). After 4 h the media was replaced with fresh media and cells were grown at 37 C for approximately 40 h. After harvesting and washing, cells were analyzed for green fluorescent protein expression using a Coulter Epics X L glow cytometer (Hialeah FL). Fluorescence was monitored at 525 nmfrom5000 cells per sample, and triplicate transfections were measured per sample.


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The supercoiled forms of the plasmid were rapidly lost by small doses of ionizing radiation. The amount of open-circle forms rose simultaneously, reached a maximum and then slowly decayed with increasing radiation exposure. Linear forms did not appear until higher doses were obtained; the amounts of these rose and reached a plateau.

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D O S E (Mrads)

Figure 2. Surviving forms of GFP-plasmid after exposure of the frozen supercoiled plasmid to various doses of ionizing radiation. Supercoiled (solid circles ), open circle (open circles ) and linear forms (solid diaminds) are shown. Normalized to total DNA originally present.

Radiation target theory predicts an exponential loss of molecules as a function of radiation exposure; the rate of this loss is directly proportional to the mass of the molecule. The supercoiled form was observed to decay in this manner. The double-stranded GFP plasmid has a mass of 3300 kDa. In three independent experiments, the amount of supercoiled DNA decreased exponentially with radiation dose (Fig. 2), yielding a target size of 3059 kDa (Table 1 ), indicating that a single radiation interaction anywhere in the plasmid



199 results in disappearance of that molecule from the supercoiled population. By itself, this does not indicate whether there are one or more product types. The plasmid preparations used in these studies contained some level (

Radiation Target Analyses of Plasmid DNA - American Chemical Society

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