Reactivity and Specificity of RNase T₁, RNase A, and RNase H toward Oligonucleotides of RNA Containing 8-Oxo-7,8-dihydroguanosine

Abstract

Understanding how oxidatively damaged RNA interacts with ribonucleases is important because of its proposed role in the development and progression of disease. Thus, understanding structural aspects of RNA containing lesions generated under oxidative stress, as well as its interactions with other biopolymers, is fundamental. We explored the reactivity of RNase A, RNase T, and RNase H toward oligonucleotides of RNA containing 8-oxo-7,8-dihydroguanosine (8oxoG). This is the first example that addresses this relationship and will be useful for understanding (1) how these RNases can be used to characterize the structural impact that this lesion has on RNA and (2) how oxidatively modified RNA may be handled intracellularly. 8-OxoG was incorporated into 10-16-mers of RNA, and its reactivity with each ribonuclease was assessed via electrophoretic analyses, circular dichroism, and the use of other C8-purine-modified analogues (8-bromoguanosine, 8-methoxyguanosine, and 8-oxoadenosine). RNase T does not recognize sites containing 8-oxoG, while RNase A recognizes and cleaves RNA at positions containing this lesion while differentiating if it is involved in H-bonding. The selectivity of RNase A followed the order C > 8-oxoG ≈ U. In addition, isothermal titration calorimetry showed that an 8-oxoG-C3'-methylphosphate derivative can inhibit RNase A activity. Cleavage patterns obtained from RNase H displayed changes in reactivity in a sequence- and concentration-dependent manner and displayed recognition at sites containing the modification in some cases. These data will aid in understanding how this modification affects reactivity with ribonucleases and will enable the characterization of global and local structural changes in oxidatively damaged RNA.

Translesion synthesis by AMV, HIV, and MMLVreverse transcriptases using RNA templates containing inosine, guanosine, and their 8-oxo-7,8-dihydropurine derivatives

“…It can be assumed that the expected H-bonding interactions between 8-oxopurines, in its syn-conformation, and A play a role in this behavior (Fig 8C). This is also in agreement with 8-oxoG exhibiting H-bonding interactions as Uridine, arising from a conformational change around the glycosidic bond, in other enzymatic contexts [53]. Interestingly, the corresponding 8-oxoI analogue also allowed for incorporation of dC and dA, with higher efficiency in the presence of dATP (app.…”

RNA from a simple-tandem repeat is required for sperm maturation and male fertility in Drosophila melanogaster

“…(Figure 1—figure supplement 3) For RNAse of embryos prior to probe hybridization: RNA-FISH to AAGAG was performed on embryos pre-treated with RNaseIII (which cleaves dsRNA; Nicholson, 2014), RNaseH (which cleaves the RNA strand in RNA/DNA hybrids), RNase I (which non-specifically cleaves ssRNA and dsRNA), and RNaseA (which cleaves adjacent to pyrimidines, preferentially in ssRNA, and specifically not between purines such as 5’-AGAAGGGAGAAG [Herbert et al, 2018; Kelemen et al, 2000]). Reaction conditions were as follows: Samples were treated in 50 μl final volume with either RNAseIII: 1X RNAseIII buffer, 1.5 μl Shortcut RNaseIII (New England Biolabs, cat.…”

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

“…To understand the impact of the modification on the structure, all strands were radiolabeled and treated with RNase A, which cleaves single‐stranded RNAs at both pyrimidine and 8‐oxoG sites into fragments containing 5′‐OH and 3′‐phosphate ends . The results were then compared with those obtained with canonical strand 1 .…”