Plasmid DNA Acquires Immunogenicity on Exposure to Singlet Oxygen

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Fozia Khan¹, Farina Khan¹, Asim A. Siddiqui², and Rashid Ali^1*

¹Department of Biochemistry, Faculty of Medicine, J. N. Medical College, A. M. U. Aligarh 202002, India; fax: (91-571) 309-5535; E-mail: foz_k76@yahoo.com

²Department of Biotechnology, All India Institute of Medical Sciences, New Delhi, 110029 India

^* To whom correspondence should be addressed.

Received February 26, 2006; Revision received March 28, 2006

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In the present study, the effect of singlet oxygen (¹O₂) (generated by ultraviolet (UV) irradiation of methylene blue) on plasmid DNA has been analyzed by UV spectroscopy, fluorescence spectroscopy, and S₁ nuclease digestibility. Both native and ¹O₂-modified plasmid DNA were treated with a number of restriction enzymes to map out the sites damaged by ¹O₂. It was also observed that, on exposure to ¹O₂, native plasmid DNA that is non-immunogenic acquired the ability to elicit an immune response in experimental animals. However, the induced antibodies exhibited appreciable cross reactivity with various polynucleotides and nucleic acids. The data indicate that the antibodies, though cross-reactive, preferentially bind ¹O₂-modified epitopes on plasmid DNA. Gel retardation assay further substantiated the enhanced recognition of ¹O₂-modified plasmid DNA over the native form. The antibodies developed were then subjected to competition ELISA with sera from various diseases such as systemic lupus erythematosus, rheumatoid arthritis, and cancer. These results suggest that upon exposure of DNA to ¹O₂, neo-epitopes are generated, which may be one of the factors for the induction of circulating autoantibodies in the three diseases.

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KEY WORDS: plasmid DNA, reactive oxygen species, singlet oxygen, autoantibodies, autoimmune disease

DOI: 10.1134/S0006297906080086

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