Oxidative damage of nucleic acids is an important event in the progression/development of disease. The C8-position in purine rings is a common target of reactive oxygen species and leads to 7,8-dihydro-8-hydroxypurines as one of the main products. We synthesized the phosphoramidite of the 8-oxoadenosine modification and used it for its incorporation into oligonucleotides of RNA via solid phase synthesis. The sequence/design for the oligonucleotides was such that the effects (structural and functional) of the lesion could be tested on different motifs. We used circular dichroism (CD) to assess the stabilization/destabilization imposed by this lesion and found that increasing the number of modifications results in depressed T<sub>m</sub> values of ca. 13-15° C per lesion (in 10-mers). Thermodynamic parameters obtained from van’t Hoff plots confirmed this pattern with increasing enthalpies and ΔΔG° values of ca. 1 kcal/mol. We also tested the effects of this lesion in short hairpins containing the tetraloop UUCX (X= A, 8-oxoA) and found that the stem is hypersensitive to substitution of A by 8-oxoA while the loop is unaffected by this exchange. In conclusion, the effects of 8-oxoA on RNA structure are position dependent and its stabilization may vary from sharp decreases to small increments, in some cases, leading to the formation of other more/less stable structures. These structural changes may have larger biological implications, particularly if the oxidatively modified RNA persists, thus leading to changes in RNA reactivity and function. We are interested in testing the effects and biological implications of this oxidative lesion on strands of known functional short RNAs e.g., miRNA, siRNA, or shRNA and how its presence may lead to changes in different biochemical pathways.

Thermal stability and functional implications of short RNA strands containing 7,8-dihydro-8-hydroxyadenosine

249th National Meeting (2015)

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