Biophysical properties, thermal stability and functional impact of 8-oxo-7,8-dihydroguanine on oligonucleotides of RNA—a study of duplex, hairpins and the aptamer for preQ₁as models

Abstract

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.

7,8‐Dihydro‐8‐oxoguanosine Lesions Inhibit the Theophylline Aptamer or Change Its Selectivity

“…To provide better understanding of the impact of 8‐oxoG as a function of position, the thermal denaturation transitions were obtained (by CD, Figure S15) to show values for aptamers 9 and 10 that are equivalent, whereas aptamers 11 and 12 have increased thermal stability. As established previously, stabilization of structure can, in some cases, be directly related to decreased affinity in target binding . The large stabilization observed in the case of aptamer 11 might be due to increased interactions between 8‐oxoG and other nucleobases, arising from extended H‐bonding networks formed from both Watson–Crick and Hoogsteen faces.…”

“…Other positions that are potential candidates for G‐to‐8‐oxoG substitution involve G14 and G29. However, two aspects deterred us from probing these positions: 1) they have not been reported to be involved in the recognition of the xanthine derivatives, and 2) given their proximity to adenosine units, they might potentially generate base‐pair interactions that stabilize the overall structure at the expense of selectivity and/or affinity …”

“…Thus, it is expected that, in a single‐stranded RNA, this will be the preferential conformation. However, because both faces can be involved in H‐bonding, this context may vary for cases in which 8‐oxoG is involved in intra‐ or intermolecular interactions, such as in its folding to other secondary structures, or in H‐bonding with other biopolymers . These factors are known to present a challenge from a biological point of view, if the modification is generated in vivo, and have been characterized in monomers and in oligonucleotides (ONs) .…”

Translesion Synthesis by MmLV-, AMV-, and HIV-Reverse Transcriptases Using RNA Templates Containing Inosine, Guanosine, and Their 8-oxo-7,8-Dihydropurine Derivatives

“…The synthesis corresponding to the phosphoramidite for 8-oxoG was carried out according to a previous report. 40 All experiments described herein were carried out in triplicate, unless otherwise noted.…”

Pseudo aptamer expands aptamer’s applications

“…Base modifications arise from chemical substitutions more work is needed to address how such changes influence base pairing, helix stability, folding and conformation. 90,91 It should not be missed obviously that the conjugation on the amine at the desired positions has been one efficient way of modifications via the simple chemical reactions, such as N-alkylation, N-coupling. These simple bases modifications can promote the pseudo aptamer properties, such as the conformations and global structures.…”