Oxidative damage of DNA has been studied to a great extent, due in part to its potential mutagenicity and toxicity. On the other hand, although the formation of oxidative lesions is as likely to occur within RNA, their effects and outcomes have been understudied in this context. Furthermore, oxidative damage of RNA has been recently linked to the development/progression of some diseases thus making the study of these processes of importance. Reactions between reactive oxygen species (ROS) and RNA can result in the formation of 8-oxo-7,8-dihydropurines (8-oxoguanosine and 8-oxoadenosine). To establish the effects of these lesions in structure and function of RNA, both modifications were independently synthesized and incorporated into oligonucleotides of RNA. To assess structural implications, we prepared RNA strands (w/wo modifications) with sequences able to form different structural motifs, i.e., duplex, single stranded, internal loops, bulges, or hairpin structures; and compared their biophysical properties to their corresponding canonical analogues. To test for functional implications, we chose the aptamers of pre-Q<sub>1</sub> and theophylline as models, due in part to their small size (ca. 34 nt long) and well established behavior. We placed the modifications at positions known to be involved in ligand binding as well as at remote sites with preliminary data showing that the presence of these lesions can result in deleterious effects rendering partial, or total, loss of function. Overall, we found that the effects of 8-oxoA and 8-oxoG on RNA structure and function depend on both position and amount of modifications. We found that their stabilization varied from sharp decreases to small increases, in some cases, leading to the formation of other more/less stable structures. Van’t Hoff plots were used to calculate thermodynamic parameters and we obtained ΔG° values of ca. 1 kcal/mol. Circular dichroism was used to determine melting temperatures values and calorimetry (ITC) was used to determine ligand binding strengths and specificities. We used the well-studied theophylline aptamer in the presence of caffeine or theobromine to detect and quantify changes in specificity. We are in the process of finishing the collection of data, which will be presented herein.

Structural effects of 8-hydroxy-7,8-dihydropurines in RNA and their effects on function using the aptamers for pre-Q<sub>1</sub> and theophylline as models

251st National Meeting (2016)

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Structural effects of 8-hydroxy-7,8-dihydropurines in RNA and their effects on function using the aptamers for pre-Q1 and theophylline as models

Structural effects of 8-hydroxy-7,8-dihydropurines in RNA and their effects on function using the aptamers for pre-Q₁ and theophylline as models