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

Abstract Aptamers are attractive constructs due to their high affinity/selectivity towards a target. Here 7,8‐dihydro‐8‐oxoguanosine (8‐oxoG) has been used, due in part to its unique H‐bonding capabilities (Watson–Crick or Hoogsteen), to expand the “RNA alphabet”. Its impact on the theophylline RNA aptamer was explored by modifying its binding pocket at positions G11, G25, or G26. Structural probing, with RNases A and T1, showed that modification at G11 leads to a drastic structural change, whereas the G25‐/G26‐modified analogues exhibited cleavage patterns similar to that of the canonical construct. The recognition properties towards three xanthine derivatives were then explored through thermophoresis. Modifying the aptamer at position G11 led to binding inhibition. Modification at G25, however, changed the selectivity towards theobromine (K d≈160 μm), with a poor affinity for theophylline (K d>1.5 mm) being observed. Overall, 8‐oxoG can have an impact on the structures of aptamers in a position‐dependent manner, leading to altered target selectivity.


Introduction
An aptameri st he smallest fragment of ab iopolymer that is able to recognize ap articulart arget with high affinity and specificity,o ften with as ub-micromolar/sub-nanomolar dissociation constant. [1] These constructs display recognition of aw ide array of different moleculesa nd their targetsr ange from large biopolymers such as proteins [2] to small molecules [3] or ions. [4] In addition, their smalls ize, high flexibility,a nd ease of manufacture make them attractive candidates for various applications, including as sensors, materials, or replacements for antibodies. [5] The potentialf or functionalization of nucleic acid aptamers with ap lethora of groups at variousp ositions has enabled the development of these systemsi nto functional structures with promise in therapeutic applications. [6] However,a potentiald isadvantage in comparison with their analogous protein structures (antibodies) is the lack of diversity in the structurals et, which is restricted to the four nucleobases (G,A , C, U). [7] This has prompted efforts to diversify the nature of the nucleobases with other groups. [8] Of note are examples in which as election process led to chemically modified aptamers with dissociation constantsi nt he nanomolar to sub-picomolar range, and with potential fort herapeutic applications,w ith selectivity towards biologically relevant targets such as cancer cell lines, aspartyl protease b-secretase 1( BACE1), proprotein convertase subtilisin/kexin type 9( PCSK9), vascular endothelial cell growth factor-165 (VEGF-165), or interferon-g (IFN-g). [9] Specifically,t he modified nucleobases that were used in these works include 7-(thiophen-2-yl)imidazo [4,5-b]pyridine, 5-chlorouracil/7-deazadATP mixtures, or C5-modified pyrimidine systems containing hydrophobic moieties such as naphthyl, phenyl, or morpholino groups. We are interested in probing the H-bonding capabilitieso fm odified nucleobases to control the functiono fa ptamersa nd to diversify the "toolkit" to generate constructs with distinct selectivity and, ideally,i ncreased affinity.
We opted to explore am odification that has been widely studied in other contexts:7 ,8-dihydro-8-oxoguanosine (8-oxoG). Of the many oxidativel esions that have been characterized in DNA or RNA, purine-derived modificationsa re expected to be the most abundant [redoxp otential trend = G < A < C < U]. [10] Twoi mportant features that make 8-oxoGaunique structuralb uilding block are:1 )its preference to undergo an anti!syn conformational change around the glycosidic bond, and 2) the distinct H-bonding patterns arising from each of these isomers( or,p ut another way,i ts ability to form stable base pairs with cytosine or adenine, Scheme1). The nature of the conformational change to the syn isomer is known to be independento fs olvent (DMSO or water)o rp H( 5-8) and is due to steric hindranceb etween the C5'-position and the atom at the C8-position. [11] Thus, it is expected that, in as inglestranded RNA, this will be the preferentialc onformation. However,b ecause 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, [12] or in H-bondingw ith other bio-Aptamers are attractive constructs due to their high affinity/selectivity towards at arget. Here 7,8-dihydro-8-oxoguanosine( 8-oxoG) has been used, due in part to its unique H-bonding capabilities (Watson-Crick or Hoogsteen), to expand the "RNA alphabet". Its impact on the theophylline RNA aptamer was explored by modifying its binding pocket at positions G11, G25, or G26. Structuralp robing,w ith RNases Aa nd T 1 ,s howedt hat modification at G11leads to adrastic structuralchange, whereas the G25-/G26-modified analoguese xhibited cleavage pat-terns similart ot hat of the canonical construct. The recognition properties towards three xanthine derivatives were then explored through thermophoresis. Modifying the aptamera t positionG 11 led to binding inhibition. Modificationa tG 25, however,c hangedt he selectivity towards theobromine (K d % 160 mm), with ap oora ffinity for theophylline (K d > 1.5 mm) being observed. Overall, 8-oxoG can have an impact on the structures of aptamersi naposition-dependent manner,l eading to altered target selectivity.
polymers. [13] These factorsa re known to present ac hallenge from abiological point of view,ifthe modification is generated in vivo, and have been characterized in monomers [14] and in oligonucleotides (ONs). [15] On the other hand, this behavior can be attractive and promising from ad esign perspective:t hat is, in the use of 8-oxoG as an ON building block to target small molecules or proteins of interest. The 8-oxoG unit offers the possibility of altered function, due to its inherent capability to generateH -bonding networks that are distinct from those generated by its canonicala nalogues,y et structurally similar.T his property has been explored in the design of compounds that fit the H-bonding pattern of 8-oxoG for its detection, [16] to control DNA structure, [17] in the formation of supramolecular helices with potential uses for electronic biodevices, [18] or in the use of C8-substituted guanosine analogues to understand biological mechanisms. [19] Of note is an example in which aD NA aptamerw as designed to detect 8-oxoG throughi nteractions similar to those shown in Scheme1. [20] In addition, the presence of 8-oxoG has also been shown to affect the overall structure of DNA and to lead to its deformation. [21] These examples provide evidencet hat, although the effects of this oxidative modification on RNA have not been explored in detail,t here is great potentiali nu sing it as ah andle to control the structure and functionofR NA.
To probe for the impact that 8-oxoGh as on the structure and function of RNA, the aptamer for theophylline was used as model.T his construct was originally selected with the development of SELEX over two decades ago. [22] Sincet hen, it has been used as am odel system for variousa pplications, [23] such as to explore new sensing methodologies, [24] to establish theoretical models, [25] or to detect theophylline in serum. [26] The construct is an RNA strand, 33 nt in length, with high selectivity for theophylline [27] (Scheme 2). This represents ag ood model because of existing detailed knowledge on the interactions between the RNA and the small molecule. Specifically,i t is known that four guanosineu nits (G4, G25, G26, G29) are involved in the recognitiono ft heophylline whereas three more are present in the vicinity of the active site (G11, G19, and G31), thus making these positions attractive candidates for probingt he impact that the 8-oxoGl esion has on structure and small-molecule recognition. This aptamer is able to recognize the smallm olecule over other targets, containing minor structurald ifferences, with up to 10 000-fold increased affini-ty. [28] It has been established, for example, that the canonical aptamer can recognize theophylline( 1,3-dimethylxanthine) over theobromine (3,7-dimethylxanthine), which differsi nt he position of as ingle methyl group,o rc affeine (1,3,7-trimethylxanthine), which contains an additional methyl group (Scheme 2A).
In brief, three levels of scaffolding provides upport for the aptamer's binding pocket (Scheme 2B), which is composed of 14 nucleotidest hat form ap latform/lowerl oop, as well as a ceiling,t hat fits the theophylline and recognizes it through hydrogen-bonding interactions with C22 and U24. The aptamer containsanS -turn andm aintainsashape that brings the lower and upper loops of the conserved regioni nto proximity to intercalate, thus generating the binding pocket. [29] Specifically, U23 H-bonds to A28, due to the S-turn in its tertiary structure, and this allows ab ase triple interaction with U6 to form the floor of the bindingp ocket,w hereas ab ase triplei nteraction between A7,C 8, and G26 provides the "ceiling" of the binding pocket. One side of the recognition site is generatedt hrough p-p stackingi nteractions between C21, A7, C22, and U6, whereas the other side is formed by G26 intercalation between G25 and U24, thought to stabilize the sharp bend in the tertiary structure to allow C22 to intercalateb etween U6 and A7.
In addition, providing furthers tabilization of the bindingc ore, A10 p-stacks with G25 andG 11.A ni mportant factor to note is that A7 is displaced from its typical A-form helical position, thus generating space that facilitates binding to the target. With regard to the high affinity towards theophylline, C22 and U24 are involved in its discrimination from other xanthine derivatives through H-bondingi nteractions with N7ÀHa nd the O6 carbonyl group of the small molecule.

Results
We set out to explore the impact of the 8-oxoG unit on the theophylline RNA aptamer and incorporated the lesion into ONs of RNA via solid-phase synthesis. The RNA construct (strand 1), 33 nucleotides in length, was modified with 8-oxoG at positions G25, G26, and G11, chosen on the basis of their roles in target recognition within the binding pocket,t oy ield RNA strands 2, 3,a nd 4 respectively ( Figure 1). Importantly,t he construct for which more structural information is available was chosen. [28] To understand the impact of the modification on the structure, all strandsw ere 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. [30] The results were then compared with those obtained with canonical strand 1.A ss hown in Figure 1( left), RNA strands 1-3 displayed hyperreactivity at positions U6 and C9, and to al ower extent some cleavage in the C20-C22 region (in the cases of modified strands 2 and 3), thus suggesting that these positions are in environments available for ribonuclease access and not restricted to H-bonding interactions with other nucleobases.C leavage at position C9 was unexpected; however,t his marks the beginningo ft he internal loop (4 1), thus presumably making it more accessible. Interestingly,incorporation of 8-oxoG at positions G25 or G26 gave rise to similar cleavage patterns, thus suggesting that modification at these sites does not affect the overall structure of the aptamer,o r that these positions are cleaved because they are also exposed in another structural arrangement. The only observed difference was that cleavage around the C20-C22r egion was increaseds lightly in the case of RNA 3 (G26-modified), thus indicating that interactions amongst nucleobases in this section of the construct might be altered. The most striking difference was observed in the case of strand 4,m odified at position G11, which displayed prominent cleavage at additional positions (C12 and C13), consistent with disruption of the stem in the upper hairpin and exposure of thesen ucleobases to the ribonuclease.A ssignmento ft he bands was carriedo ut by comparing the results against ah ydrolysis ladder( NaHCO 3 ,p H9.1). The ladderw orks through cleavage of every nucleobase from the 3'-end, thus allowing ab and to appear at every positiono f the aptamer. Ap attern in whichd oublet bands are observed in cases of shorter fragments is consistent with the formation of the 3'-phosphate, along with the corresponding cyclic phosphate derivative that has been characterized/observed previously. [31] To complementt hese observations, experiments were also carriedo ut with RNase T1, ar ibonuclease that specifically cleavesa ta ll G-sites in as ingle-stranded context. Interestingly, the only aptamer that showeds ome hypersensitivity to this ribonuclease was aptamer 4,w hich displayed cleavage at positions G14, G18, and G19. Consistent with the resultso btained in the case of the RNase Ac leavage, this observation points to disruption of the upper hairpin within the construct. Although the intensity of the cleavage bands was weak, the pattern was reproducible. Furthermore, in agreementw ith alteration at the binding site, cleavage at position G29 took place in the case of RNA strand 2,w hereas position G25 (G26 wasr uled out because of the reported inability of RNase T1 to cleave at 8-oxoG sites) was accessible for ribonuclease activity in the case of modified aptamer 3.
In an attemptt oi nterpret these changes and to explain the reactivity patterns, the UNAFold server was used to explore possible changes in structure (Figures S5-S8 in the Supporting Information). [32] As expected,t he model for the canonicalR NA 1 matched relatively well with the known structure, with the only exception being the prediction of an A11:U24 base pair ( Figure S5). Because 8-oxoGi sn ot available in this hybridization package, we reasoned that substituting the modification with uridine could provideavalid model, on the basis of the H-bondings imilarities between syn-8-oxoGa nd U. However, this approach predicted structural changes in the case of RNA strands 2/3 (modified at G25 and G26) that do not explain the observedR Nase Ac leavage patterns ( Figures S6 and S7). The same analysis was carriedo ut by substituting G11w ith U, which resulted in disruption of the hairpin stem in this region but did not fully explain the RNase Ac leavage pattern at positions C12 and C13. Overall,s ubstitution of Ui np lace of 8-oxoG did not provideagood model in this context.
To assess the impact that 8-oxoG might have in the upper stem, RNA strands 5 and 6,c ontaining Go r8 -oxoG (Scheme 3), were prepared in order to mimic this structural motif. The substitution wasi ncorporateda tp osition2 of this construct and all strandsd isplayed bands consistentw ithf ormation of ah airpin (bands with positive and negative ellipticity at 260 and 210 nm, respectively). Thermal denaturation transitions wereo btained by measuring the hypochromic shift in the dichroic signal at 260 nm as af unctiono ft emperature and were independentofconcentration, thus suggesting unimolecular transitions. Incorporation of 8-oxoG, as in strand 6,r esulted in al arge thermald estabilization relative to canonical RNA hairpin 5.T his is consistent with other cases in which an oxidative lesion has alarge impact on hairpin stability. [33] To confirm that the 8-oxoGb ase pairs with Ci nt his context, hairpins 7 (containing U) and 8 (lacking an ucleotide)w ere also prepared, and displayedl arger thermald estabilization in both cases.T his result indicates that 8-oxoG does interact with Ca tt his position, albeit more weakly than in the case of the correspondingW Cp air.T he observed trends were in agreement with those predicted by use of UNAFOLD, albeit with values that wereconsistently lower than those observed experimentally (Figures S9-S11a nd Scheme3,i nset). Overall,t he differencei nt he thermal stability between hairpins 5 and 6 (DT m %À9 8C) suggestst hat positioning 8-oxoG in the aptamer at this position (G11, Figure 1) leads to ad rastic structural change, yet to be fully characterized.

Small-molecule binding
We then explored the impact that the 8-oxoG lesion has on small-molecule recognition and used microscale thermophoresis (MST) to establish the selectivity anda ffinity of each construct towards the three xanthine derivatives. This technique was chosen because it allows for the determinationo ft he dissociation constant (K d )b etween an aptamer and its cognate ligand in free solution and with minimal sample consumption. [34] This technique relies on recording the thermophoretic effect of the aptamer in the absence and in the presence of the target molecule, [35] and is carriedo ut by measuring changes in fluorescence as the bound/unbound ON migrates acrossaheat gradient inside ac apillary. [36] To this end, ONs (modified at the same positions as in the previouss trands: G11, G25, G26) containing ac yanine-5 dye (l abs = 646 nm, l em = 662 nm) at the 3'-end were prepared via solid-phase synthesis to yield RNA strands 9-12 ( Figure 2A). To interpret the data from the binding thermophoretic assays, the recommendations from ar ecent report were followed. [37] In addition, because interactions between nucleic acids and their targets can be affected by salt and buffer concentrations [38] we used buffer systemsthat have been reported previously. [28b] Binding checks were performed in the presenceo fe ach targett ov alidate interactions between the small moleculea nd the aptamer, or to rule out aptamerst hat do not display specific binding;these experiments were carried out by measuring the ON mobility either in the absence or in the presence of high concentrations of the small molecules. As depicted in Figure 2B (left) the canonical aptamer 9 displayed binding to the expected target, theophylline, whereas no bindingo ccurred in the presence of theobromine or caffeine. Solutions containing the small molecule were then prepared by making sixteen twofold dilutions, to obtain ad issociation constant (K d )o f (29 AE 35) mm,avalue consistent with the literature. [28b, 29] In addition, isothermal titration calorimetry (ITC) wasu sed to validate binding of the canonical RNA aptamer 1 to theophylline, to obtain a K d value of 1.35 mm ( Figure S24). Interestingly, modified aptamers 10 and 11 also displayed binding affinity towardst heophylline, albeit with affinitiesa pproximately two orders of magnitude weaker (K d > 1.5 mm,F igure 2D). An exact quantity could not be established, due to the low solubility of the small molecules in the buffer system at higherc oncentrations. The fact that these two aptamersrecognize theophylline, to some extent, is not surprising in view of their similars tructural characteristics (as observed through enzymatic cleavage experiments). On the other hand, as shown in Figure 2C,a pta-mer 10 (modified at G25) displayed ah igherb inding affinity towardst heobromine, with K d > 160 mm (a complete sigmoidal curve forthis pair could not be obtained, due to the poor solubility of theobromine), and with no binding of caffeine.T his result highlights the ability of 8-oxoG to have an impact on structure as well as on small-molecule affinity and selectivity. Furthermore, consistent with large structuralc hanges, aptamer 12 (modified at G11) did not displaya ny binding affinity towards the three xanthine derivativest ested in this work. To provideb etter understanding of the impact of 8-oxoG as a functiono fp osition, the thermald enaturation 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 thermals tability.A se stablished previously,s tabilization of structure can, in some cases, be directly related to decreased affinity in target binding. [12] 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-bondingn etworks formed from both Watson-Crick and Hoogsteen faces.
Attempts to record changes in structure by CD in the absence and in the presence of the xanthine derivativesd id not show any appreciable differences ( Figure S23). All aptamers displayed dichroic bands consistent with folding into A-form duplexes.A lthough the structuralp robinge xperiments indicate that ad ifferent structure might be formed in the cases of RNAs 4/12 (modified at positionG11), the CD spectra showed that the extento ft he duplex region is comparable to those of the other modified RNA strands, as well as that of the canonical aptamer ( Figure S15). Figure 2. A) Sequences of Cy-5-labeled RNAs 9-12 andtheir corresponding thermal denaturation transitions (T m ), obtained in a1 0mm sodium phosphate buffer (pH 7.5)a nd a50mm Tris·HCl saline (TBS) buffer (pH 7.6). MST bindingcheck, binding traces, and K d -fit curve of B) construct 9 with theophylline, and C) construct 10 with theobromine (additionalcurves are included in Figure S14,d isplayingt he same bindingc heck and K d calculation with ad ifferent buffer). The blue trace corresponds to unbound RNA, the green trace to RNA boundt ot he small molecule, and the red traces correspondt othe RNA titratedw ith the small molecule (somec urvesa td ifferent concentrations were omitted for clarity). D) K d -fit curve of constructs 10/11 with theophylline and 9 (no binding) with theobromine. All curves wereo btained in triplicate.

Discussion
The impact of 7,8-dihydro-8-oxoguanosine (8-oxoG)o nR NA structurea nd function was explored by using the aptamer for theophylline as am odel.T he 8-oxoG motif was independently incorporatedo nto ONs of RNA and its structural impactw as assessed by electrophoretic analyses and circular dichroism, whereas its functionw as established by assessingt he ability of the modified aptamerst ob ind three differentx anthine derivatives:t heophylline, theobromine, and caffeine.T hermophoretic analyses showed that the selectivityw as alteredi nt he case of modified aptamer 8 (8-oxoG at position25), which displayed K d values in the micromolar range for theobromine and in the millimolar range for theophylline.
The results from the RNase A/T1 cleavage studies, along with results obtained from MST experiments,i ndicatet hat a modification at G11h as the most drastic impact on the aptamer's structure, whereas modificationsa tG 25 and G26 alter the structures lightly and still allow binding of theophylline. Al-thoughR NA 2/10 has features that suggest as tructure similar to that of the canonicala nalogue 1/9,b ased on enzymatic degradation and T m analyses, ac hange in the H-bonding pattern of 8-oxoG25 might induce ac hange in the binding pocket. It is plausible that the anti-to-syn flip for 8-oxoG25 allows it to hydrogen bond with A10, in turn allowing the formation of WC base pair G26:C9, breaking interactions with C8, which ultimately disruptst he ceiling for the theophylline binding pocket (Scheme4A). C8 is necessary for the canonical aptamer'sh igh-affinity interaction with theophylline. The 8-oxoG25 and A10 base pairing also disrupts the p-stacking interaction between G25 and A10;t his could then destabilize the binding core and might displace conserved residues such as C21 and C22. The large increase in K d is also consistent with ad isruption in the binding pocket.T he slight degradation seen at G25c ould indicatet hat the H-bond to A10 is relatively weak. Furthermore, the highera ffinity towards theobromine can be explained by thed isplacemento fC 22, which is integral in discriminating for theophylline. The conservation of U24 does not provide the ability to discriminate theophylline from xanthined erivatives because N9 is the same in both theophylline and theobromine, andt his could thus explain why this aptamerc an bind to theobromine.
The resultso btainedw ith RNAs 3/11,m odified at position 26, showed an increaseo fo ver 100-fold in K d towards theophylline, and al ack of recognition for theobromine or caffeine. It is possible that, as depicted in Scheme 4B,i nteractions with U6, which is pivotal in forming the floor of the binding pocket, are disrupted by the expected 8-oxoG26:A7 base pair. This could also displace or preventU6'sinteraction with its corresponding upper-loop residues and impart stability,e vident from al arger T m (74.5 8C). As mentioned above,A 7n eeds the ability to move out of the A-form helix to create space for the small molecule, and this is unachievable if it is H-bonding with 8-oxoG26. This distinct H-bond might also aid in the formation of an ew ceiling for the small molecule, because G25 can now H-bond with C8. The H-bond between the 8-oxoG and A7 might also disrupt the p-stacking interactions of C21, A7, C22, and U6 by abolishing its ability to intercalate between C21 and C22;t his could decrease the interaction between the upper and lower loop residues. Both RNAs 2/10 and 3/11 retain the ability to bind to theophylline, althoughw eakly,b ecause U24 is stillc onserved in the core and is potentially not affected by adverseH -bonding or p-stacking interactions, while still having an impact on the binding pocket.
We initially hypothesized that modification at G11( strands 4/12)w ould not greatly impact small-molecule recognition. However,t his RNA did not bind to anyo ft he xanthined erivatives. This can be explained in terms of the large structural changes observed in the upper loop, evident from the distinct RNase A/T1 cleavage pattern ands uggesting that 8-oxoG11 disrupts the upper scaffolding of the aptamer overall. It is possible that 8-oxoG11c ould be involved in H-bond interactions with A16 or A17, which would explain why both G18 and G19 are exposed to degradation.I na ddition, this could result in C21 and C22 being prevented from intercalatingw ith A7 and U6, in turn affecting the role of U24 within the binding core and intercalationb etween G26 and A28, which would ultimately destabilize the S-turn that is needed to maintain the integrity of the binding pocket. Attempts to explain the overall structure with the assistance of the UNAFold enginew ere carried out by substituting 8-oxoG with U; however,the predicted structures, as well as other possibilities (Scheme 4C), do not match well with the cleavage patterns. Overall, although the structures of the G11-modified aptamersw ere not established, this suggests that the presence of 8-oxoG at this positionh as al arge impact on the structure and functiono ft his aptamer. Other positions that are potentialc andidates for G-to-8-oxoG substitution involveG 14 and G29. However,t wo aspects deterred us from probing these positions:1 )they have not been reported to be involved in the recognition of the xanthine derivatives, [29] 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. [12] Lastly,a nother point of relevance relates to the biological impact that oxidative stress has on RNA. Oxidized RNA has been shown to be presenti nv arioust ypeso fR NA, including rRNA, [39] mRNA, [40] or miRNA, [41] and this also makesr iboswitches [42] prone to oxidative damage. Althought his relationship has not been established, it is plausible that such processes might play ar ole in dysfunction or alteredr egulatory mechanisms. Thus, understandingt he potentials tructural/functional changes arising from the formation of oxidative lesions is of importance to assesst heir biological implications.

Conclusion
The impact that 8-oxoG has on structure and function, through small-molecule recognition, was probedw ith use of the theophyllinea ptamera samodel. It was shown that the effect of a single 8-oxoG modification varies as af unctiono fp ositiona nd that it can result in decreased, or abolished, affinities towards the cognate target molecule, or that it can also change the selectivity of the aptamert owards ad ifferent target. Specifically, modification at G25 led to preferential binding of theobromine over theophylline. This result suggests that 8-oxoG might be usable as am odification in the discovery of aptamersw ith distinct selectivity/affinity.A lthough this is ap romising strategy, obstacles remain to overcome, including 1) the development of sequencingt echnologies that would enable as election process that includes 8-oxoG in RNA (although recent advances show promise in this respect), [43] and 2) deeper understanding of the impact of 8-oxoG on RNA structures within various structuralm otifs-aspects that are not trivial and that we are workingt oaddress.

Experimental Section
General:8 -OxoG phosphoramidite was synthesized out according to ap revious report. [12] All experiments were carried out in triplicate.
RNA synthesis:O Ns were synthesized with a3 94 ABI DNA/RNA synthesizer and use of CPG supports and 2'-O-TBDMS phosphoramidites (purchased from Glen Research). 5-Ethylsulfanyl-1H-tetrazole (0.25 m)i na cetonitrile was used as the coupling reagent, dichloroacetic acid in dichloromethane (3 %) was used for deblocking, a2 ,6-dimethylpyridine/acetic anhydride solution was used for capping, and an iodine/THF/pyridine solution was used in the oxidation step. Coupling times of 10 min were used. ONs were deacetylated/debenzoylated/deformylated and cleaved from the CPG support in the presence of 1:1a q. methylamine (40 %) and aq. ammonia (40 %) with heating (60 8C, 1.5 h). Am ixture of 1-methylpyr-rolidin-2-one/triethylamine/HF (3:2:1) was used for removal of the TBDMS groups (60 8C, 1h), followed by purification by electrophoresis (20 %d enaturing PAGE). C18-Sep-Pak cartridges were obtained from Waters and used to desalt the purified oligomers with NH 4 OAc (5 mm)a st he elution buffer.O Ns were dissolved in H 2 O and used as obtained for subsequent experiments. Unmodified ONs were purchased from IDT-DNA or ChemGenes and, after quantification by UV/Vis, used without further purification. ONs containing af luorescent Cy-5 probe at the 3'-end were synthesized on resin (purchased from Glen Research) containing this moiety and purified as described above.
UV/Vis spectroscopy:C oncentrations of all ONs were determined by UV/Vis with aP erkinElmer l-650 UV/Vis spectrometer and quartz cuvettes (1 cm pathlength). General UV/Vis spectra were also taken with a1mm pathlength and 1 mLv olumes (Thermo Scientific Nano Drop Nd-1000 UV/Vis spectrometer). Origin 9.1 was used to plot and normalize spectra of monomers and ONs for comparison.
Circular dichroism (CD) spectroscopy and thermal denaturation transitions (T m ):C Ds pectra were recorded at various temperatures (PTC-348W1 Peltier thermostat) with use of quartz cuvettes with a 1cmp athlength. Spectra were averaged over three scans (325-200 nm, 0.5 nm intervals, 1nmb andwidth, 1s response time) and background-corrected with the appropriate buffer or solvent. Solutions containing the RNA strands had the following compositions: RNA (1.5 mm), MgCl 2 (5 mm), NaCl (10 mm), sodium phosphate [pH 7.3 (or other pH values whenever appropriate), 1mm]. All solutions that had been prepared in order to record thermal denaturation transitions (T m )w ere hybridized prior to the recording of spectra by heating to 90 8Cf ollowed by slow cooling to room temperature. T m values were recorded at 270 nm with ar amp of 1 8Cmin À1 and step size of 0.2 with temperature ranges from 4t o9 5 8C. A thin layer of mineral oil was added on top of each solution to keep concentrations constant at higher temperatures. Origin 9.1 was used to determine all T m values and to plot CD spectra of RNAs with/without small molecules.
ON labeling:T 4p olynucleotide kinase (PNK) and g-32 P-ATP-5'-triphosphate were obtained from PerkinElmer.O Ns were labeled by mixing PNK, PNK buffer,A TP,D NA, and water (final volume 50 mL) according to the manufacturer's procedure followed by incubation at 37 8Cf or 45 min. Radiolabeled materials were passed through a G-25 Sephadex column followed by purification by electrophoresis (20 %d enaturing PAGE). The bands of interest (slowest) were extruded and eluted over as aline buffer solution (0.1 m NaCl) for 36 hat378C. The remaining solution was filtered and concentrated to dryness under reduced pressure followed by precipitation over NaOAc and ethanol. Supernatant was removed and the remaining ON was concentrated under reduced pressure and dissolved in Microscale thermophoresis (MST):T his technique was used to establish binding affinities and potential for binding with the three xanthine derivatives. RNA strands modified with the Cy-5 fluorophore at the 3'-end were used.
MST-small-molecule binding studies:T he small-molecule binding studies were performed with 16 twofold dilutions of each small molecule as can be seen in Ta ble 1. The goal was to measure binding at varying concentrations of small molecule with ac onstant concentration of RNA to determine the corresponding dissociation constants (K d ). The Monolith NT.115 MST system was used for these studies with concentrations of small molecules as shown in Ta ble 1. Theobromine did not readily dissolve in the buffers used here and was dissolved in DMSO (100 %). This solution was then diluted in the corresponding buffer to achieve solutions with a DMSO content lower than 5%.P recipitated theobromine was observed in the cases of solutions in which the small molecule was present in the 5mm range, so experiments were set such that the concentration of theobromine was 1mm maximum.
Binding checks were accomplished for all RNAs with each small molecule prior to K d determination. The binding checks were carried out to validate binding of the small molecule and aptamer. Eight samples were prepared-four that contained the RNA aptamer and four that contained the RNA and the small molecule. Both the RNA and the small molecule were kept constant at 10 nm and at the highest small-molecule concentration (e.g.,t he theophylline concentration in the presence of RNA 9 was 11.25 mm). The prepared mixtures were incubated at room temperature for % 10 min, captured in ac apillary tube (as described below), and placed on the plate, with the RNA sample in rows 1-4 and the RNA/small molecule complex in rows 5-8.
Dissociation constants were obtained as follows. First, 16 microtubes (0.6 mL) were filled with 10 mLo ft he buffer containing the appropriate small molecule. For theophylline and caffeine, the buffer was 1 TBS [pH 7.5, Tris·HCl (50 mm), NaCl (150 mm)], whereas for theobromine the buffer was 1 TBS with DMSO (10 %). This was carried out in such am anner that sequential twofold dilution, with respect to the small molecule, was present in each tube. This was followed by addition of RNA (20 nm,previously denatured and hybridized by heating to 90 8Ca nd slow cooling to RT,1 0mL) to each tube to accomplish 10 nm [RNA] (while keeping the content of DMSO at 5% DMSO max.). The mixtures were then incubated in ice for 20 min in the cases of the mixtures containing theophylline or caffeine and at room temperature in those of the mixtures containing theobromine. Next, % 10 mLo ft he mixture was captured in ac apillary tube, placed on the plate, and covered with the magnetic strip to prevent movement. Each mixture was withdrawn and handled one at at ime, with the solutions containing the highest concentration of small molecule placed at position "1". The microtube and the capillary were held horizontally to prevent bubble formation, and the capillary was handled by the end of the tube. Each programmed experiment ran for approximately 20 min.

RNA structural probing
RNase A:Acocktail solution of RNA (3000-5000 counts) in phosphate buffer (pH 5.5, 10 mm)w as made. Am ixture of the RNA and the enzyme (1:1) was prepared and incubated at RT for 1h.A fter incubation, loading buffer [LB:f ormamide (90 %), EDTA( 1mm), 7 mL] was added, and the mixture of interest (9-10 mL) was added to ad enaturing PAGE. For shorter oligomers (< 20 nt) as hort gel was used. For longer oligomers al ong gel was used. The gels were run until the methylene blue dye ran halfway to three quarters of the way down the gel.
RNase T 1 :Ac ocktail solution of RNA (3000-5000 counts) in phosphate buffer (pH 5.5, 10 mm)w as made. Am ixture of the RNA and the enzyme (1:1) was prepared and incubated at 50 8Cf or 45 min. After incubation, LB (7 mL) was added, and the mixture of interest (9-10 mL) was loaded onto ad enaturing PAGE. For shorter oligomers (< 20) as hort gel was used. For longer oligomers (> 20) a long gel was used. The gels were run until the methylene blue dye ran halfway to three quarters of the way down the gel.
The degradation patterns for all aptamers were compared against ah ydrolysis ladder (0.5 m NaHCO 3 ,p H9.1), which was used for band assignment. The ladder works through hydrolysis of every nucleobase from the 3'-end and was produced by incubating the RNA of interest and the hydrolysis buffer at 90 8Cf or 12 min (68:32 RNA/NaHCO 3 ratio, by volume) followed by addition of loading buffer (formamide, 90 %) prior to loading onto gel. mics Facility at Colorado State University.I na ddition, characterization of ONs 5-8 was carried out at the University of Colorado's Bruker Center forE xcellence (Departmento fP harmaceutical Sciences, Skaggs Schoolo fP harmacy and PharmaceuticalS ciences, The University of ColoradoA nschutz Medical Campus), partially funded by the L.S. SkaggsP rofessorship and NIH grant R35GM128690, under the guidanceo fJ ustin Jens (laboratory of Prof. Vanessa V. Phelan) This work was based, in part, on aM .S. thesis presented and defendedb yC .K. at CU Denver.T his work was supported by an ORS grant from the University of Colorado Denver.F unds from NSF-MRI-1726947 facilitated this work. Funding from NIGMS (1R15GM132816) is also acknowledged. ITC and MST measurements were carried out at the Biophysics core facilities, Structural Biology and Biochemistry-University of Colorado Anschutz MedicalC ampus. We would like to thank Shaun Bevers for helpful discussions and aid in thed esign of these experiments.

Conflict of Interest
The authors declare no conflict of interest.