Oxidative stress may result in the formation of the C8-modified guanosine 8-hydroxy-7,8-dihydroguanosine(8-oxoG). This lesion is thought to be involved in the development/progression of neurodegeneration. However, its effect on the structure and function of different RNA secondary structures is still relatively unknown. Herein we report on the independent synthesis and incorporation of 8-oxoG into oligonucleotides of RNA to address its effects with respect to structure and function. The lesion was placed in different locations on strands able to form different structural motifs, namely duplexes, hairpins, and pseudoknots. Circular dichroism was used to obtain melting temperatures (Tm) and determine thermodynamic parameters. We observed a trend in which increasing the number of 8-oxoG lesions decreased their corresponding Tm values. This is consistent with an induced instability within the strands. The same trend is expected to be seen on hairpins. To address the effects of this lesion on function, we used the well studied preQ1 and theophylline aptamers as models. Isothermal titration calorimetry was used to establish Kd values of 0.50 μM and 0.72 μM for the preQ1 and theophylline aptamers, respectively. The incorporation of 8-oxoG lesions into each aptamer should destabilize the RNA structure and lower its ability to bind selectively. We are in the process of testing ligand specificity using caffeine and theobromine as substitutes of theophylline. In conclusion, RNA strands that contain 8-oxoG lead to structural instability, thus resulting in adverse effects on their function.

Effect of 8-hydroxy-7,8-dihydroguanosine on the structure and function of RNA hairpins and aptamers of preQ1 and theophylline

251st National Meeting (2016)

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