Aptamers are known to regulate various cellular metabolic and biochemical pathways. Reactions between reactive oxygen species and adenosine are known to produce the oxidative lesion, 8-oxo-adenosine (8-oxo-A). This modification is reported to form base-pair mismatches (8-oxo-A:G instead of A:U) that may inhibit formation of their intended (canonical) structure and/or induce changes in secondary structure. Thus, if present in aptamers of RNA, functional variations can be expected. This phenomenon was tested by conducting structural studies of RNA strands containing the modified lesion. We used sequences corresponding to aptamers with recognition for queuosine or aminoacridine. These aptamers were chosen based on their length (short, 15-34 nt long) and known behavior. The modified monomer was obtained via a 3-step reaction starting with bromination and acetylation of the C8-position followed by hydrolysis in the presence of ammonia to yield the desired lesion in ca. 53% yield. The oxidized nucleotide was then protected at the N6- and C5’-positions by treatment with N,N-dimethylformamide dimethyl acetal followed by tritylation, respectively. Silylation of the C2’ position was achieved using standard conditions in the presence of tert-butyldimethylsilyl chloride and silver nitrate. Phosphitylation of this material yielded the desired phosphoramidite in a ca. 41% yield (over 4 steps) of 8-oxo-A for its incorporation into oligonucleotides of RNA via solid phase synthesis. Each synthetic intermediate was characterized via NMR, IR, UV-Vis, and MS, while the identity of each oligonucleotide was confirmed via MALDI-TOF MS. Structural analysis were carried out using CD and PAGE. We found that the effects of 8-oxo-A were position dependent ranging from decreased activity to complete loss of functionality. In conclusion, oxidative modifications in adenosine may lead to deleterious effects in the function of RNA aptamers.

Structural changes induced by 8-Oxo-7,8-dihydroadenosine in RNA oligonucleotides may result in loss of function of RNA aptamers

249th National Meeting (2015)

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