8-Oxo-7,8-dihydropurines as Building Blocks to Enhance the Selectivity of an RNA Aptamer for Aminoglycosides

The use of nucleic acids as potential therapeutic tools, sensors, or biomaterials, among other applications, has dramatically increased. Among these, RNA aptamers are of interest due to an innate high specificity toward their cognate targets, which include small molecules, proteins, ions, or cells. In this work, we took advantage of the ability that 8-oxo-7,8-dihydroguanine (8-oxoG) has to participate in unique H-bonding interactions, and probed its use to increase/control the selectivity/affinity of aptamers of RNA and DNA. The chosen model is a 23-nt long RNA (Neo61/Neo1–5′-GGA CUG GGC GAG AAG UUU AGU CC) strand that folds into a pentaloop hairpin with a stretch of three G·U Wobble pairs within the stem, which is known to have affinity toward various aminoglycosides. 8-OxoG was incorporated at positions G6, G7, G10, G12, or G15, within aptamers composed of RNA, DNA, or 2’-OMe modified RNA. Their recognition was tested toward 9 small molecule targets with aminoglycoside (x8) or antibiotic (x1) backbones, and their affinities were measured via circular dichroism (CD). Isothermal titration calorimetry (ITC) was used to corroborate the use of CD as a reliable technique. It was determined that incorporation of 8-oxodG at position-12 within DNA (OG12-DNA) led to increased selectivity toward neomycin or ribostamycin (K_d ≈ 2.5 and 2.2 μM), displaying 1–2 orders of magnitude tighter binding compared to other targets. Furthermore, functionalization with 8-oxodG at position-6 (OG6-DNA) displayed increased selectivity toward neomycin or tobramycin, albeit with decreased affinities (K_d ≈ 46 and 53 μM). Interestingly, the canonical DNA aptamer also displayed 4–10 fold enhanced selectivity toward neomycin, ribostamycin, and gentamicin, compared to its RNA homologue. On the other hand, the corresponding RNA analogues containing 8-oxoG or other modifications, specifically 8-oxoinosine, inosine, 8-oxoadenosine, or uridine, resulted in a high level of promiscuity toward most aminoglycosides, with kanamycin and streptomycin generally exhibiting higher dissociation constants. The presence of 2’-OMe-modified ribose led to trends similar to those obtained with their corresponding canonical RNA constructs. From a structural perspective, all nucleobase modifications led to thermal destabilization of the aptamer (including the DNA analogues), while the presence of the 2’-OMe ribose modification resulted in increased thermal stability. Among the molecules tested, neomycin and ribostamycin induced significant structural changes (measured via CD) on aptamers of RNA or DNA. Changes in RNA included the formation of a new band with positive ellipticity (λ_max ∼ 285 nm), associated with glycosyl bond rotation along the G·U wobble pairs that presumably facilitates recognition. On the other hand, binding by canonical and OG12 DNA aptamers resulted in a B-to-A form transition, where the smaller major groove may serve to facilitate DNA-target interaction. Further structural data were obtained by carrying out structural probing assays in the presence of RNase A, T₁, or DNase I; which displayed varying degradation patterns and thus changes in secondary structure as a function of the position of 8-oxoG/8-oxodG and presence/absence of the small-molecule target. Overall, the results reported herein show that (1) the use of 8-oxodG within DNA increases aptamer selectivity toward neomycin and/or ribostamycin; (2) the presence of 8-oxoguanine can alter the function of RNA and DNA, which is of broad biological relevance; and (3) the introduction of 2’-OMe modifications does not affect the selectivity of the aptamers in this work. While it is early to predict how 8-oxoG will affect the selectivity of aptamers at large, this work provides a link between the structure and function of oxidized RNA.