Biophysical properties thermal stability and functional impact of 8-oxo-78-dihydroguanine on oligonucleotides of RNA—a study of duplex hairpins and the aptamer for preQ1 as models

摘要

A better understanding of the effects that oxidative lesions have on RNA is of importance to understand their role in the development/progression of disease. 8-oxo-7,8-dihydroguanine was incorporated into RNA to understand its structural and functional impact on RNA:RNA and RNA:DNA duplexes, hairpins and pseudoknots. One to three modifications were incorporated into dodecamers of RNA [AAGAGGGAUGAC] resulting in thermal destabilization (ΔTm – 10°C per lesion). Hairpins with tetraloops c-UUCG*-g* (>8-10), a-ACCG-g* (>11-12), c-UUG*G*-g* (>13-16) and c-ACG*G*-g* (>17-20) were modified and used to determine thermal stabilities, concluding that: (i) modifying the stem leads to destabilization unless adenosine is the opposing basepair of 8-oxoGua; (ii) modification at the loop is position- and sequence-dependent and varies from slight stabilization to large destabilization, in some cases leading to formation of other secondary structures (hairpin→duplex). Functional effects were established using the aptamer for preQ1 as model. Modification at G5 disrupted the stem P1 and inhibited recognition of the target molecule 7-methylamino-7-deazaguanine (preQ1). Modifying G11 results in increased thermal stability, albeit with a Kd 4-fold larger than its canonical analog. These studies show the capability of 8-oxoG to affect structure and function of RNA, resulting in distinct outcomes as a function of number and position of the lesion.