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Reactivity and Specificity of RNase T1, RNase A, and RNase H Towards Oligonucleotides of RNA Containing 8-Oxo-7,8-dihydroguanosine Cassandra Herbert, Yannick Kokouvi Dzowo, Anthony Urban, Courtney N Kiggins, and Marino J.E. Resendiz Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00277 • Publication Date (Web): 23 Apr 2018 Downloaded from http://pubs.acs.org on April 24, 2018

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Reactivity and Specificity of RNase T1, RNase A, and RNase H Towards Oligonucleotides of RNA Containing 8-Oxo-7,8-dihydroguanosine Cassandra Herbert, Yannick Kokouvi Dzowo,† Anthony Urban,† Courtney N. Kiggins, and Marino J. E. Resendiz* Department of Chemistry, University of Colorado Denver, Science Building 1151 Arapahoe St, Denver, CO 80204, USA * To whom correspondence should be addressed. Tel: 303-315-7658 ; Email: [email protected]

These authors contributed in the same amount to this work.

ABSTRACT Understanding how oxidatively damaged RNA interacts with ribonucleases is of importance due to its proposed role in the development/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 T1, and RNase H towards oligonucleotides of RNA containing 8-oxo-7,8-dihydroguanosine (8oxoG). This is the first example that addresses this relationship and will be useful towards 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 10-16mers of RNA and its reactivity with each ribonuclease was assessed via electrophoretic analyses, circular dichroism, and through the use of other C8-purine modified analogs (8-bromoguanosine / 8methoxyguanosine / 8-oxoadenosine). RNase T1 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 1

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sequence and concentration dependent manner, and displayed recognition at sites containing the modification in some cases. These data will aid to understand how this modification affects reactivity with ribonucleases and will enable the characterization of global and local structural changes in oxidatively damaged RNA. INTRODUCTION The phenomenon of oxidative damage to RNA has been gaining attention due to its potential role in nature, e.g., in the development/progression of disease(1-2), on protein synthesis (3), or as a signaling pathway in plants (4). In our efforts to understand the impact that oxidative damage has on RNA structure, function, and interactions with other biopolymers, we decided to explore the reactivity between RNases and RNA containing, arguably, the most relevant oxidative lesion in RNA/DNA, 8oxo-7,8-dihydroguanine (8-oxoG). Our motivation was driven by 1) the role of ribonucleases in probing of RNA structure to gain information about its function (5); and 2) work highlighting cellular mechanisms designed to cope with oxidative stress and their relationship to processes that lead to disease or cellular dysfunction, specifically those involving oxidized RNAs (6). Ribonucleases (RNases) carry out essential functions in RNA metabolism (7) and the discovery of biomolecules with distinct reactivity and roles is of interest, e.g., a recent example includes an enzyme that specifically targets A/G sites (8). Since damage of RNA can pose a threat to the integrity of biological pathways (9), it is important to identify how cells cope with oxidative stress and degrade RNAs, a process that is well established in some cases and not well understood in many others, e.g., for micro-RNAs (10). Furthermore, the role of ribonucleases in structural probing has been a common and robust approach to assess conformational and structural changes for over three decades (11-12). Interestingly, the formation of 8-oxoG has also been used as an intermediate to establish some structural parameters in RNA, thus highlighting its uniqueness as a reactive probe (13). Modification of the C8-position within purine rings in DNA and RNA results in conformational changes that lead to altered H-bonding patterns. It is well established that the presence of a functional group, or atom larger than hydrogen, at the C8-position leads to rotation 2

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around the glycosidic bond towards the syn-conformation. This conformational change exposes a different set of H-bonding interactions (Scheme 1) and may result in the formation of base pair mismatches, specifically 8-oxoG:A (14-15) or 8-oxoA:G (16), or altered participation with other biopolymersleading to the formation of crosslinks (17). Examples where enzymes can recognize oxidized nucleotides (18), RNA containing 8-oxoG (19-20), or other modifications (21), have been reported and highlight the ability of these molecules in such processes. In addition, instances where enzymatic function is altered by the presence of 8-oxoG have been attributed to changes in H-bonding (22) or structural conformation (23).

Scheme 1. All types of RNA are subject to processing or degradation, and numerous cellular mechanisms are involved or affected by these important actions (24), therefore, modification of RNA arising from oxidative stress may prove to be a challenge to the RNA machinery. In fact, a relationship between oxidative stress and RNA processing has been reported in the context of endonucleolytic cleavage of tRNA (25) or in monitoring by APE1 in ribosome biogenesis (26). We are interested in the use of ribonucleases to gain information on the local and global structural aspects of RNA containing modifications generated from oxidative stress and used three ribonucleases for our initial studies: RNase T1, A, and H (RNase H1, E. coli rnhA gene product). These RNases were used because of their common use in structural probing, distinct reactivity, and commercial availability. RNase T1 is a microbial ribonuclease that cleaves at the 3’-end of sites containing a guanine nucleobase in single-stranded RNA (27); RNase A is a distributive (binds→cleaves→releases) endoribonuclease that binds to the nucleobases of RNA and catalyzes the cleavage of a 3’-phosphate bond of nucleotides containing a pyrimidine ring (28); and RNase H belongs to a class of 3

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endonucleases that specifically cleave RNA in RNA:DNA duplexes to yield 5’-phosphates and 3’-OH ends (29). Interestingly, an association between RNase H enzymes and the formation of 8-oxoG within RNA has been proposed (along with the ability of RNase H2 to cleave a C-site when opposing 8-oxoG (30)) and is an area that promises interesting discoveries in the near future (31) thus requiring a closer look at these interactions. This article represents part of our initial efforts to address the structural and functional implications that oxidatively generated lesions have on RNA. We chose oligonucleotides 10-16 nucleotides (nt) long taking into consideration that short RNAs such as microRNAs are also prone to oxidation (32), and because of the relatively easier assignments and reactivity assessment, compared to longer oligonucleotides that have the ability to fold into various secondary structures. The reported findings will enhance our understanding on the interactions between ribonucleases and RNA containing 8-oxoG, 8-oxoA, and other C8-modified nucleobases. Furthermore, we are in the process of using the obtained information to examine how these oxidative modifications change the overall structure of strands of RNA with other secondary structures, via structural probing that takes advantage of the reactivity described herein. MATERIAL AND METHODS General Methods. General methodology in the synthesis of phosphoramidites and modified oligonucleotides is similar to that previously reported (33). All canonical ONs were purchased from IDT-DNA. All experiments were carried out in triplicate. CD Spectroscopy. CD spectra were recorded at various temperatures (PTC-348W1 peltier thermostat) using Quartz cuvettes with a 1 cm path length. Spectra were averaged over three scans (350-200 nm, 0.5 nm intervals, 1 nm bandwidth, 1 s response time) and background corrected with the appropriate buffer or solvent. All solutions prepared to obtain thermal denaturation transitions (Tm) were hybridized prior to recording spectra by heating to 90 °C, followed by slow cooling to room temperature. Melting temperatures were recorded at 270 nm with a ramp of 1 °C / min and step size 4

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of 0.2 with temperature ranges from 4 °C to 95 °C. 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 thermal denaturation transition values. Experiments carried out to obtain Tm values from RNase A and RNase T1 were prepared in 10 mM sodium phosphate buffer, pH 5.5, while those to address RNase H experiments were carried out in RNase H buffer (75 mM KCl, 50 mM Tris-HCl, 3mM MgCl2, 10 mM dithiothreitol, pH 8.3, 1x reaction buffer from manufacturer). Spectra to determine secondary structure from this buffer displayed a poor signal to noise ratio at wavelengths below ca. 240 nm, therefore structural aspects and some Tm values were obtained in buffered solutions (10 mM sodium phosphate buffer, 5 mM MgCl2, pH 5.5). Oligonucleotide labeling. T4 polynucleotide kinase (PNK) and γ-32P-ATP-5′-triphosphate were obtained from Perkin Elmer. Oligonucleotides were labeled by mixing PNK, PNK buffer, ATP, RNA, and water (final volume = 50 µL) according to manufacturer’s procedure followed by heating to 37° C for 45 min. ONs containing either 8-bromo or 8-methoxy modifications (ONs 5, 6, 10) resulted in large amounts of degradation, thus they were incubated at room temperature. Radiolabeled materials were passed through a G-25 sephadex column followed by purification via electrophoresis (20 % denaturing PAGE). The bands of interest (slowest) were excised and eluted over a phosphate saline buffer solution (10 mM NaCl, 10 mM Na2P2O7, pH 7.2) for 6 h at 37 °C. 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 oligonucleotide was dried and re-dissolved in water. Activity was assessed using a Beckmann LS 6500 scintillation counter. Degradation with RNases. RNase A and RNase T1 were obtained from Thermo Scientific and added to water (4 µL into 46 µL H2O) to obtain the first dilution (Table 1). Subsequent 10-fold dilutions were made each time to obtain solutions with lower enzyme concentrations and are labeled as a dilution factor Z in the text and in the supporting materials. Specifically, a Z factor of 2 implies a 100-fold dilution and a Z factor of 3 refers to a 1000-fold dilution (See Table 1 for enzyme amounts used per experiment). Fresh solutions were prepared for each experiment every time. The desired 5

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ribonuclease concentration was mixed in a 1:1 ratio with the RNA of interest (3,000-5,000 counts) followed by incubation for 45 min (rt for RNase A and 55 °C for RNase T1), and diluted with loading buffer (90 % formamide) before loading onto the gel. RNase H (1 µL, obtained from New England Biolabs) was added to water (39 µL) containing 1X RNase H buffer (7.5 mM KCl, 5 mM Tris-HCl, 0.3 mM MgCl2, 1 mM dithiothreitol, provided by manufacturer). Subsequent 10-fold dilutions were made each time to obtain solutions with lower enzyme concentrations. As described above, a Z dilution factor of 2 refers to a 100-fold dilution (Table 1). The desired ribonuclease concentration was mixed in a 1:1 ratio with the RNA:DNA duplex of interest (3,000-5,000 counts) followed by incubation for 3.5 h at 37 °C and subsequent addition of loading buffer, prior to loading onto gel. RNA:DNA duplexes were prepared in RNase H buffer and the samples were placed in a heat block (90 °C) followed by slow cooling to rt over ca. 2 h. These solutions contained the RNA (ca. 3,000-5,000 counts) and DNA (ca. 100-fold excess) in 1x RNase H buffer.

Table 1. Amounts of RNase used per experiment. The text describes Z or 10Z as a dilution factor in the figures. Hydrolysis Ladder. γ-32P-5′-Oligonucleotide of interest is diluted with water (7.5 µL total volume), typically containing 3-4 times as many counts as oligonucleotide in the experiment, followed by addition of hydrolysis buffer (3.5 µL 500 mM NaHCO3, 10 mM EDTA, pH 9.1), and heated to 90 °C 6

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for 20 min. The mixture is briefly placed on a micro-centrifuge followed by addition of loading buffer (90 % formamide) before loading onto the gel. Isothermal Titration Calorimetry. Measurements were acquired on a MicroCal iTC200 microcalorimeter (Microcal, Inc.) at 25 °C with an enzyme (RNase A) concentration of 60 µM (determined via UV-vis) and small molecule concentration of 0.9 mM. Both the ligand and small molecule were dissolved in a buffer consisting of 5 mM NaOAc, pH 5.5 with 0.33 % DMSO. RNase A was dialysed in the same buffer prior to use and concentrations were determined via UV-vis spectroscopy. The small molecule (P1-P4) was titrated into the sample cell in 2 µL injections, with a reference power of 10 µcal s-1, initial delay of 600 s, 180 s spacing, and a stirring speed of 750 rpm. Analysis of the data was performed using Origin 7.0 ITC software (Microcal Software Inc) via fitting to a single-site binding model. Concentrations for the solutions of the nucleotides were established via UV-vis by comparing to known extinction coefficients (34-35). RESULTS Reactions with RNase T1. To initiate our studies, we decided to use a ribonuclease that specifically hydrolyses single-stranded RNA at G-sites (RNase T1). Reactions were carried out on dodecamers of RNA containing five Guanosine units (0-3 8-oxoG modifications, ONs 1-4) in the presence of RNase T1 in buffered solutions (sodium phosphate, pH 5.5 at 50°C). The size and sequence were chosen based on previous reports suggesting that strands as short as 4 nt-long can be substrates of this endonuclease (36). As expected, cleavage of the canonical oligonucleotide displayed products that were consistent with fragmentation at each guanosine unit with reactions occurring in ca. 50 % overall yield (combined products). Interestingly, and contrary to our initial hypothesis (vide infra), the ribonuclease did not recognize oxidatively generated damaged sites and bypassed positions where an 8-oxoG is present. This is evident from the lack of 1, 2, or 3 bands for ONs 2, 3, or 4 with respect to the canonical model ON 1 (Figure 1). This observation suggests that the H-bonding interactions within the binding pocket of the ribonuclease are disrupted by the presence of the N7-H along with other possible interactions arising from the C=O-8 position. To further explore this hypothesis, taking 7

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into consideration that 2’- or 3’-phosphates of 8-BrG (known to prefer the syn-conformation) can inhibit ribonuclease activity (37-38), we decided to incorporate this modification to yield ON 5. In addition, guanosine was functionalized at the C8-position with a methoxy group followed by its incorporation into RNA to yield ON 6. Phosphoramidites for the 8-BrG and 8-OMeG derivatives were obtained according to previous reports (39-40). However, we were surprised to observe that, within the context of the dodecamers used, neither of these C8-modifications functioned as substrates for RNase T1. The same results were observed at higher enzyme concentration, varying buffers, or varying temperature. Thus, it is reasonable to conclude that although the hydrogen bonding patterns present in 8-oxoG may play a role in the lack of recognition by RNase T1, the group at the C8position (in this case an oxygen) may exert a more important influence in this respect.

Figure 1. 20% Denaturing PAGE of RNA 1-4 following treatment with RNase T1 (experiment carried out in triplicate, Fig S1). ONs 5 and 6 also displayed a lack of reactivity towards this RNase (Fig S2). [RNase] – Z = 5, refer to Table 1. Reactions with RNase A. We then decided to test the reactivity of strands 1-4 towards a ribonuclease with different specificity, RNase A (targets pyrimidine containing sites in single stranded RNA), in part encouraged by a report suggesting that the nucleotide phosphate of 8-oxoG can 8

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function as substrate within the binding site of this enzyme (41). It was hypothesized for 8-oxoG to be a likely target, given the conformational change around the glycosidic bond towards the syn-isomer, which potentially forms H-bond networks that mimic those of a pyrimidine ring (8-oxoG:A base pair mismatches mimic the H-bonding pattern of uridine). Gratifyingly and in agreement with our hypothesis, a distinct cleavage pattern was observed on oligonucleotides that contained between 1-3 oxidatively generated modifications that suggested recognition of 8-oxoG by the RNase A. As illustrated in figure 2, one cleavage band was observed on strand 1 while strand 2, containing one 8oxoG modification, displayed a band at this position. As expected, strands 3 and 4 displayed two or three bands respectively, consistent with the presence of additional 8-oxoG sites. To gain more information on the selectivity of the enzyme within this dodecamer, experiments were carried out while varying the concentration of the ribonuclease. It was observed that a 108-fold dilution resulted in cleavage at the pyrimidine site (U9) exclusively (Figure S3). This suggests that the cleavage displayed at 106 dilution (Figure 2, left) may be initiated via ribonuclease activity occurring from multiple cleavage events. This change in selectivity suggests a mechanism that is due to slow association/fast dissociation from the oligonucleotide and that RNase A may preferentially bind to pyrimidine rings over 8-oxoG sites with one caveat, that the cleavage can be dependent on the nature of the 5’-nucleotide with better cleavage in the order A>G>C>U (42) thus preventing an accurate conclusion from direct comparison using this sequence.

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Figure 2. 20% Denaturing PAGE of RNA 1-4 following treatment with RNase A at dilution factors of Z = 6, or 7 (Table 1) Experiments carried out at Z = 2-8 RNase A are included in the supporting materials Fig S3. To further assess the reactivity of the ribonuclease and establish its selectivity, dodecamers containing a single pyrimidine or 8-oxoG lesion were synthesized and treated with RNase A (Figure 3, ONs 7-10). The sequence was chosen to avoid potential secondary structures while examining the specificity of the reaction in a controlled manner (only one cleavage site). The reactivity was also explored in the context of position by varying the location of the 8-oxoG modification, closer to the 5’-end (ON 11). Consistent with the reactivity observed on the original constructs (ONs 1-4), one cleavage band was observed on ONs containing U, C, or 8-oxoG, while being inert towards a sequence containing no pyrimidine rings (ON 7). Experiments carried out at higher enzyme concentrations did not have an impact on the lack of reactivity on ON 7. Interestingly, varying the concentration of the ribonuclease showed a difference in reactivity that depended on the nature and position of the nucleobase and a higher efficiency towards cleaving C-sites was observed. This result is in contrast to ON 9 containing uracil where a 10-fold higher concentration in enzyme was necessary for recognition of the ON containing a U-site, consistent with previous trends (43). Similarly,

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cleavage of ONs 10 and 11, containing 8-oxoG, required higher concentrations of RNase A and displayed similar reactivity as that of the ON containing U (Figure 3).

Figure 3. 20% Denaturing PAGE of RNA 7-11 following treatment with RNase at varying enzyme concentrations. Z corresponds to the order of magnitude in the dilution factor, e.g., 102 = 100 folddilution, see Table 1. To gain insights on RNA-enzyme H-bonding interactions and explore on the impact arising from the presence of the oxygen at the C8-position, oligonucleotides modified with 8-BrG (ON 5) and 8-OMeG (ON 6) were treated with RNase A. Experiments were carried out and compared to canonical and 8-oxoG modified strands 1 and 2 at an enzyme concentration that ensured reactivity at an 8-oxoG site (Figure 4A). As shown, one cleavage band was observed in each case except for ON 2, which contains 8-oxoG and displayed two fragments, with one band corresponding to hydrolysis at the 8-oxoG site. To corroborate this lack of reactivity, experiments were carried out at various concentrations, where no change was observed (Figure S5) unless the enzyme concentration was increased significantly. In cases where the dilution factor for RNase A was between 1 and 10 x, a band that indicated recognition of the 8-OMe functionalized guanosine unit in ON 6 was observed in yields < 10 % (Figure S6). The reactivity of the C8-OMe analog was corroborated by preparing 11

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analogs of ON 10, functionalized with one 8-BrG or 8-OMeG to yield ONs 12 and 13 respectively. Experiments carried out on these strands of RNA also displayed a lack of reactivity with the 8-BrG analog, along with the appearance of a weak band in the case of the 8-OMe strand (Figure S7). These observations suggested that the N7-H is necessary for recognition and that the oxygen at the C8position is playing and important role within the binding site. To gather more information we carried out experiments on decamers of RNA containing 8-oxoA, where the sequence was chosen based on a previous report for which the RNA strand contains between 0-3 modifications (ONs 14-17) (44). The expectation was that 8-oxoA had the structural features to serve as substrate of the ribonuclease, based on previous reports showing that phosphate nucleotide analogs can act as inhibitors of this enzyme (45-46). However, experiments carried out on these ONs displayed a lack of reactivity towards 8oxoA (Figure 4B) and only cleavage bands at positions C9 and U2 were observed. The results suggest that while the H-bonding patterns with resemblance to U or 8-oxoG are present (C=O and NH) and can potentially bind to Thr45, an essential residue in the binding site (vide infra), the exocyclic amine is potentially generating adverse interactions that prevent binding and subsequent cleavage.

Figure 4. 20% Denaturing PAGE of RNAs 1, 2, 5, and 6 (A); and 14-17 (B); following treatment with RNase A at varying enzyme concentrations. Z corresponds to the dilution factor, e.g., Z = 3 is a 1,000 X dilution (see Table 1 for exact amounts).

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One additional piece of information was obtained upon carrying out the reaction at different pH values (pH 3-9) (47). In agreement with previous reports, experiments at pH 3 did not show cleavage and experiments carried out between 5 and 7 displayed similar results (with the latter showing less effective cleavage overall). However, reactions carried out at pH 9.2 resulted in increased specificity towards sites containing uridine, moderate increased specificity towards sites containing 8-oxoG, and no effect towards sites containing cytidine. An observation that is consistent with deprotonation of the N3- or N7-positions in U or 8-oxoG, respectively, that resembles the Hbonding pattern observed in C. Furthermore, the lack of an effect upon increased pH values in samples containing C can be explained from the lack of acidic protons within the pyrimidine ring. These reactions were carried out in the absence of divalent cation Mg2+ to avoid non-enzymatic degradation, via a transesterification mechanism, (48-49) or RNase inhibition in the presence of this ion. We then decided to test the ability of RNase A to detect single point mutations and carried out the corresponding experiments on RNA:RNA duplex samples 7:18, 8:18, 9:18, 10:18, and 11:18 containing G, 8-oxoG, U, or C at the site of interest (Figure 5). It is known that this ribonuclease has the ability to recognize several base pair mismatches, including C•C and C•U (50). Gratifyingly, the corresponding experiments displayed cleavage on duplexes containing these mismatches while duplexes that contained an 8-oxoG:C base pair did not function as substrates for enzymatic cleavage. This is an important finding because it provides a way to identify the structural context in the vicinity of an 8-oxoG lesion, that is if 8-oxoG is involved in H-bonding interactions. This reactivity opens the possibility for the use of RNase A to interrogate oxidatively damaged RNAs and establish the effects of the lesion within different structural contexts to assess its local environment. Electrophoretic analyses using non-denaturing PAGE showed the formation of duplex structures for all samples prior to treatment with the enzyme. To ensure that duplex samples were formed in each case, under the conditions optimal for RNase A activity, the corresponding thermal denaturation transitions and CD spectra were obtained. In agreement with duplex formation, CD spectra displayed all the features of an A-form duplex and Tm values obtained for each sample displayed transitions well above room 13

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temperature (incubation temperature for RNaseA). In an attempt to increase the stability of duplexes 8:18 and 9:18, experiments were carried out in the presence of MgCl2, however, the presence of this divalent salt impacted the ability of RNase A to cleave the ONs in an efficient manner.

Figure 5. Treatment of duplexes of RNA w/wo base pair mismatches G:C, C:C. U:C, and 8-oxoG:C at a single site followed by treatment with RNase A along with recorded Tm values via circular dichroism ([RNA] = 3 µM, 10 mM sodium phosphate – pH 5.5). [RNase] – Z = 6 for 7/8 and Z = 5 for 9/10/11. In light of the observed ability for RNase A to recognize 8-oxoG, we set out to explore its potential as an inhibitor of this enzyme. We took advantage of reported procedures that use isothermal titration calorimetry (ITC) to measure the inhibitory activity of this ribonuclease with the corresponding 2’- or 3’-phosphate nucleotides of C (51-52). The 2’-methyl phosphate derivatives of C, U, G, and 8-oxoG P1 – P4 were obtained (Figure 6) as described in the supporting information, and used to explore the interactions between each small-molecule and the enzyme. The methyl derivatives of each phosphate were used due to the ease in purification (via silica gel chromatography of all precursor derivatives) while potentially retaining the properties needed for binding. Gratifyingly, experiments carried out on the C analog P1 (50 mM NaOAc, pH 5.5) validated the use of these models and led to isotherms that were consistent of an exothermic reaction (data corrected for heat of dilution), albeit with Kd values ca. 4-fold larger (ca. 20 µM) than those previously reported. A discrepancy that can be rationalized from the presence of the methyl group, as steric hindrance is

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increased. Experiments carried out in the presence of the 8-oxoG methylphosphate P4 displayed a binding isotherm with a dissociation constant that was within error of that observed for the C analog. Unexpectedly, the G-methylphosphate also displayed an isotherm consistent with binding at similar affinities to that observed with 8-oxoG, while the corresponding U-derivative P2 did not display any binding activity. Experiments aimed at affecting the ability of the enzyme to cleave the target ONs were carried out by introducing the phosphates into cocktail solutions containing the RNase A/ON mixture. However no effect was observed in concentration ranges up to 10,000-fold in excess of the small molecule with respect to that of control ON 8, which suggests that the ONs function as better substrates than the small molecules.

Figure 6. Structure of methyl monophosphate derivatives P1-P4 and their corresponding thermograms obtained via ITC. Reactions with RNase H. The enzyme that was used is commercially available and its source is from an E. coli strain. In general, the cleavage mode for this type of ribonucleases is dependent on the nature of the RNase H, length of DNA and RNA, and sequence (53). Knowing that tridecamers and dodecamers can be substrates for RNase H cleavage (54-55), and that a 6-8-mer of DNA is sufficient to promote RNase H activation (56) we set out to explore the reactivity of RNase H with 15

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dodecamers 1, 2, 5, 6 and 7, 10, 12, 13 by hybridizing with their corresponding DNA complements 19 or 20. Formation of duplexes was corroborated via native PAGE and circular dichroism with recorded thermal denaturation transitions above 37 °C, temperature recommended for incubation with this ribonuclease. In agreement with previous results, incorporation of 8-oxoG led to depressed Tm values with a difference of ca. -10 °C, a value that was comparable to that obtained for 8bromoguanosine (destabilizing effect of RNA duplexes containing 8-BrG has been reported) (39). We decided to compare RNase H activity towards the canonical duplex 1:19 with respect to its modified analogs 2:19, 5:19, and 6:19, with a modification at position-G6 (Figure 7A) at varying RNase H concentrations. The cleavage pattern on the canonical model (1:19) resulted in major cleavage at positions G5-G7 at higher enzyme concentrations, while lower [enzyme] led to hydrolysis around positions G6-U9. In contrast, the presence of 8-oxoG (ON 1:19) resulted in major cleavage at position U9 at the highest [RNase H] used. The only exception was observed at high nuclease concentrations, where cleavage at 8-oxoG sites was observed, albeit with lower efficiencies. The similarity in the cleavage patterns at high concentrations can be explained from multiple cleavage events on the same RNA molecule, which potentially abolishes the effect of 8-oxoG on the shorter fragments. Therefore, it can be concluded that the presence of 8-oxoG affects the activity of the enzyme significantly (at lower [RNase H]), presumably due to the structural disruption within the Aform duplex arising from 8-oxoG. To gather more information on this observation, the same reaction was carried out with duplexes 5:19 and 6:19, which contain an 8-bromoguanosine or 8methoxyguanosine at the site of interest, and that resulted in a major reactive site at position G5 or U9 at high or low [RNase H] respectively. These results suggest that the modification at this site alters the cleavage ability of RNase H.

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Figure 7. Treatment of RNA:DNA duplexes after treatment with RNase H displays major and minor cleavages, represented by the size of the arrow. Dotted arrows (green) depict cleavage sites at lower ribonuclease concentrations. Tm values are also shown and were acquired via circular dichroism recorded at 270 nm ([RNA] = 2 µM, RNase H buffer final volume = 7.5 mM KCl, 5 mM Tris-HCl, 0.3 mM MgCl2, 1 mM dithiothreitol [provided by manufacturer]). Electrophoresis was carried out on 17

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20 % denaturing PAGE for all experiments. Z corresponds to the dilution factor, e.g., Z = 2 is a 100 X dilution as detailed in Table 1. (A) Shows reactivity towards RNA:DNA duplexes containing ONs 1, 2, 5, 6 and (B) 7, 10, 12, 13 (Gel shown on Figure S8). To corroborate on the activity of RNase H and explore the possibility of assessing a general trend, the same experiments were carried out on oligonucleotides 7:20, 10:20, 12:20, and 13:20 (with 8-oxoG, 8-BrG, or 8-OMeG positioned at G6) (Figure 7B). Interestingly, a similar pattern was observed for the canonical duplex (7:20), compared to double stranded sample 1:17, where major cleavage bands were observed at positions G6-G7 at high [RNase H]. Lower enzyme concentrations displayed a different cleavage pattern at positions G6-A8 and A11. Analysis of the degradation patterns of the modified duplex 10:20 resulted in a shift in reactivity at low [RNase H] with major cleavage occurring away from the modification site at positions A10 and A11. On the other hand, increasing [RNase H] led to a major cleavage band at the modified site (X6) along with a minor band at G5. Moreover, experiments carried out on 12:20 and 13:20 displayed the same reactivity and showed a major hydrolysis band at the modification site in high [RNase H] or closer the 3’-end (A11/A10) under low enzyme concentrations. These observations indicate that degradation patterns shift away from the modification site (under low [RNase H]), however cleavage at modified site is preferred at high [RNase H]. We then carried out experiments on longer ONs (16-mers) with the goal of obtaining more information on the ribonuclease activity while attempting to find general trends. In this regard, we chose a sequence with known activity towards RNase H (ON 21, Figure 8) and that is derived from a region surrounding the TAR RNA loop bulge structure (trans-activating region, involved in nascent viral transcripts) (57) and decided to incorporate one modification near the reported cleavage sites (ON 22 and 23) or away from these positions (ON 24). However different overall degradation patterns were observed, possibly due to slight changes in the buffer conditions (authors added DMSO) or the presence of fluorescent dyes (FAM/DAB). This work will discuss two significant, but different, results that were obtained from treatment of RNA:DNA duplexes of these strands at varying 18

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concentrations of RNase H. We used the DNA strand that was reported (ON 25) and that is 2-nt longer than the RNA complement. Thermal denaturation transitions were measured in the presence of the RNase H buffer, which led to values well above the incubation time of the reactions (37 °C). Experiments carried out at higher RNase H concentrations led to the same degradation pattern in the canonical RNA (21:25) as well as duplexes 22:25 / 23:25, with major cleavage bands at positions G6 / C7 and minor bands at positions A5 / C8. However, a distinct pattern was observed for RNA 24:25 with a shift towards one major cleavage site (U9) and one minor site at G4, consistent with ribonuclease activity away from the vicinity of the site containing the 8-oxoG modification. On the other hand, reactions carried out at lower ribonuclease concentrations led to distinct degradation patterns in each case, where the canonical duplex 21:25 showed major (G14, G10, U9) and minor (A13) bands closer to the 3’-end and to the modifications in ONs 20 and 21. Therefore RNase H induced cleavage on ON 22:25 displayed the same pattern as the canonical analog (ON 21:25), with the exception of position 14, which contains the 8-oxoG site. Furthermore, reactions carried out on ON 23:25 displayed a new band at position (C15) along with the two major bands obtained on the other model ONs (G10 and U9). Lastly, treatment of ON 24:25 with RNase H resulted in the appearance of a minor band at position A13 along with the band that was also present at higher RNase H concentration (U9). All band assignments were carried out via side-by-side comparison with samples treated with hydrolysis buffer using 20 % denaturing PAGE.

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Figure 8. Treatment of RNA:DNA duplexes 21-24 after treatment with RNase H displays major and minor cleavages, represented by the size of the arrow. Tm values are also shown and were acquired via circular dichroism recorded at 270 nm ([RNA] = 2 µM, RNase H buffer final volume = 7.5 mM KCl, 5 mM Tris-HCl, 0.3 mM MgCl2, 1 mM dithiothreitol [provided by manufacturer]). Z corresponds to the dilution factor, e.g., Z = 2 is a 100 X as described in Table 1. Electrophoresis was carried out on 20 % denaturing PAGE for all experiments. We also decided to test the reactivity of the 16-mers against RNase T1 and A. Gratifyingly, in agreement with our previous observations, experiments carried out on single stranded RNAs 21-24 in the presence of RNase T1 resulted in cleavage at every G-site and bypassed locations where an 8oxoG is present (Figure S9). We also carried out experiments on these ONs using RNase A with the expectation that this ribonuclease would lead to additional cleavage at sites containing an 8-oxoG, however, the same cleavage pattern was observed in each case. That is, bands corresponding to two bands corresponding to C7 and C8 along with other fragments at U3 and C2. No new bands were observed for ONs 22-24 with respect to canonical model 21, which was surprising given our observed results on dodecamers. To explain this result we examined the possible formation of secondary structures via CD and determined that ONs 21 and 23 display the features that are common of 20

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secondary structure, appearance of a band with negative ellipticity at 210 nm along with a hyperdichroic shift in the band at 270 nm. A survey of the possible structural motifs that are thermodynamically stable with this sequence (in the absence of the DNA complement) displayed hairpin 25 as the best option, with a calculated Tm value of ca. 59.8 °C (58). Interestingly, the CD results are in agreement with this structure as the presence of 8-oxoG at positions G6 or G14 would be expected to disrupt the stem and lead to a lack of folding, while substitution at position G12 does not affect the structural motif (Figure 9). Of particular note is the significantly higher Tm value corresponding to ON 23, compared to that of ON 21, explained from potential H-bonding interactions between 8-oxoG and other nucleobases within the proposed loop, in turn inhibiting reactivity with RNase A. While the structural details arising from the presence of 8-oxoG within hairpin loops has not been explored in detail, stabilization has been reported in tetraloops and pseudoknots (33). Although no secondary structure was observed on RNAs 22 or 24 (established via CD from the absence of a band at ca. 210 nm), their lack of reactivity towards enzymatic cleavage at positions G6 or G14 may be explained from the possible formation of a range of other structural motifs in which 8oxoG participates via H-bonding interactions.

Figure 9. Calculated secondary structure of RNAs 21 and 25 along with their corresponding thermal denaturation transitions, obtained via CD (10 mM Sodium phosphate buffer, pH 5.5). ‘2o struct’ describes formation of a secondary structure, via CD, that is folding of coil into the proposed hairpin. DISCUSSION The phosphoramidite of 8-oxoG was used to incorporate this oxidatively generated lesion into oligonucleotides of RNA 10-16 nucleotides long. Ribonucleases commonly used in structural probing and with implications in some biological processes were used to explore the reactivity between RNase 21

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A / RNase T1 / RNase H and the modified RNAs. Overall, it was found that 8-oxoG is recognized by RNase A while RNase T1 bypasses sites with the lesion. RNase H displayed a distinct reactivity that depended on the sequence, generally this ribonuclease also bypassed sites containing this modification at low [RNase]. Three additional C8-modified purines were synthesized and incorporated into ONs of RNA of varying sequence to aid in explaining the nature of the reactivity between oxidatively damaged RNA and these ribonucleases, namely 8-bromoguanosine, 8-methoxyguanosine, and 8oxoadenosine. In addition, four methyl phosphate derivatives (P1-P4) were synthesized and used as potential inhibitors of RNase A to observe that the 8-oxoG analog functions as substrate of the enzyme. RNase T1. The crystal structure of RNase T1 containing guanosine monophosphate in the binding site shows that the nucleobase adopts the syn conformation with various H-bonding contacts directing the corresponding recognition (Figure 10, G-model was built from information in: 59-60). Three reasons led us to rationalize that 8-oxoG could be a substrate for RNase T1: (1) that 8-oxoG is known to exist in the syn-conformation preferentially; (2) that the H-bonding sites present in G are still part of those in 8-oxoG; and (3) that the N1 and O6 positions, known to interact with the enzyme (61), are not modified in the 8-oxoG oxidative lesion. However, contrary to our hypothesis, RNase T1 did not recognize 8-oxoG sites or C8-modified guanosine units, namely 8-BrG and 8-OMeG (Figure 10 middle and right). These observations indicate that while the presence of the N7-H may introduce a counterproductive interaction with the NH around Asn-43, the presence of the oxygen atom, or other functional groups at the C8-position, is more likely to cause the observed lack of recognition. Possible steric interactions between the group at the C8-position and the amino acid stretch Asn43/Tyr-42/Lys-41, in the vicinity of the ligand, may be responsible for cleavage inhibition. This reasoning can be corroborated upon close analysis of the crystal structure (62-64) (Figure 10, top left), where the spacing around the C8-position appears to be a limiting factor.

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Figure 10. Known H-bonding interactions involved in the specificity of the enzyme (bottom right) and proposed structures that explain the lack of reactivity of 8-oxoG sites and C8-functionalized guanine rings (bottom left and top right). The image on the bottom right was obtained from the Protein Data Bank (RCSB PDB), as referenced in the text. RNase A. The recognition of 8-oxoG by RNase A and its similarity in reactivity with U can be rationalized if one examines the H-bonding interactions between these nucleobases and the enzyme, where the contacts of 8-oxoG are similar to those observed in Uridine. Figure 11 displays the similarities in H-bonding upon comparing uridine and 8-oxoG as well as the differences with C, where the exocyclic amine is present and thus provides support for the observed results (the model for C and U was reproduced, in part from ref. 65). It is reasonable to assume, based on conformational transformations that change the H-bonding patterns, that the 8-oxoG lesion will interact with the enzyme in a manner that is reminiscent of uracil-containing sites. Although the preference by RNase A to cleave pyrimidine rings over purine rings has been suggested to be based on steric hindrance, the fact that 8-oxoG can be a substrate indicates that H-bonding patterns may play a more important role and favor this modification (as it is known to exist in its syn- conformation). As shown in figure 11, 23

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the crystal structure of RNase A with 3’-CMP as ligand (66) shows that there are no space constraints that would avoid the purine ring to fit within the binding pocket. Interestingly, substitution of the C=O with a bromine resulted in lack of recognition of the 8-BrG as substrate, presumably due to the lack of interactions with threonine-45. While introduction of a methoxy group leads to cleavage, albeit with very low efficiency. It is possible that the oxygen in the methoxy group is involved in Hbond interactions that may facilitate its recognition while the methyl group places steric hindrance that governs the diminished reactivity. On these grounds, we decided to explore the role of the C6C=O position and incorporated 8-oxoA, however it was not recognized by the enzyme hence indicating that the exocyclic amine may be disrupting the H-bonding pattern arising from the carbonyl at positions C6 and C4 in 8-oxoG or Uridine respectively. The proposed models should prove useful in the design/characterization of enzymes that specifically recognize 8-oxoG in RNA.

Figure 11. Proposed models that explain the reactivity of 8-oxoG and lack thereof in the 8-OMeG, 8BrG, and 8-oxoA cases. The image on the left was obtained from the Protein Data Bank (RCSB PDB), ref. 63-64; and a pyrimidine ring was drawn to illustrate that space constraints may not be a factor in fitting a purine nucleobase within the binding site; T45 is also highlighted. RNase H. Overall, it was shown that the cleavage patterns of this enzyme are largely affected by the presence of the 8-oxoG modification in a concentration and sequence dependent manner. Furthermore, these changes can be different from those induced by other functional groups, specifically bromo or methoxy, which suggests that very small structural variations can alter 24

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ribonuclease activity. It is likely that these changes occur from differences in thermal stabilities, where the 8-oxoG does not disrupt the RNA:DNA helix as much as the other two modifications used herein. Previous examples have shown that the presence of small modifications can result in significant changes in reactivity with some reports of note: 1) loss of activity of RNase H has been reported upon substitution of RNA with 2’-Fluoro substituents (known to exist in the 3’-endo conformation), attributed to electronic and geometric differences (67); and 2) it has also been shown that cleavage is shifted if the DNA strand contains oxidatively induced modifications (68); or that the presence of ethyl groups along the backbone of the 20-mer used shifts the degradation patterns away from these sites (69). Importantly, these examples also used RNase H from E. coli and point to the importance of the structural aspects within the RNA:DNA duplexes to maintain fidelity, efficiency, and selectivity of this ubiquitous ribonuclease. It will be of interest to see how these changes in reactivity may affect the downstream pathways of various RNAs, should they be affected by modification under oxidative stress. This information may also be useful in assessing the role of RNase H in nature, suggested to be involved in the evolution of the viral genome (70). In conclusion, the use of the ribonucleases explored herein can be useful in determining the global and local changes imposed by the ubiquitous oxidatively generated modification, 8-oxo-7,8dihydroguanine (8-oxoG). Furthermore, it provides valuable information as to how the presence of this lesion alters the interactions between both biopolymers and results in altered cleavage patterns that may affect the biological function of RNA if affected by oxidative stress. Thus, while in some cases the presence of this modification may lead to increased rates of degradation and affect specificity of the enzyme, other cases may shift cleavage patterns with yet to be discovered effects. ASSOCIATED CONTENT Supporting Information The supporting information is available free of charge on the ACS Publications website at DOI: XXXX. Details corresponding to the synthesis of the modified oligonucleotides (ONs) of RNA (pp. 25

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S4-S5), characterization of all ONs (via MALDI-TOF, Figure S10-S15), and electrophoresis as described throughout the text are also included in the supporting materials. CD spectra and Tm measurements of all the relevant structures (Figures S16-S35). All experimental details and spectra corresponding to the synthesis of P1-P4 are included in the supporting information (pp. S25-S50). ACKNOWLEDGEMENT C.H. and Y.K.D. would like to acknowledge an Undergraduate Research Program (UROP, CU Denver) for support. We thank the Department of Chemistry at Metro State University for facilitating the use of their NMR facilities. We thank the University of Colorado Denver for support in the purchase of the CD spectrometer. Mass Spectrometry of oligonucleotides was carried out at the Mass spectrometry core facilities, University of Colorado Skaggs School of Pharmacy and Pharmaceutical Sciences, Anschutz Medical Campus. ITC measurements were carried out at the Biophysics core facilities, Structural Biology and Biochemistry -University of Colorado Anschutz Medical Campus and would like to thank Shaun Bevers for helpful discussions and aid in the design of these experiments. FUNDING This work was supported via start-up funds and a CLAS Research Innovation Seed Program grant from the University of Colorado Denver. REFERENCES (1) Küpfer, P. A., and Leumann, C. J. (2014) Oxidative damage on RNA nucleobases. in Chemical Biology of Nucleic Acids, RNA Technologies, DOI 10.1007/978-3-642-54452-1_5, pp. 75-94 Springer-Verlag Berlin Heidelberg. (2) Poulsen, H. E., Specht, E.; Broedbaek, K., Henriksen, T.; Ellervik, C., Mandrup-Poulsen, T., Tonnesen, M., Nielsen, P. E., Andersen, H. U., and Weimann, A. (2012) RNA modifications by oxidation: A novel disease mechanism? Free Rad. Biol. Med. 52, 1353-1361.

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(14) Thiviyanathan, V., Somasunderam, A., Hazra, T. K., Mitra, S., and Gorenstein, D. G. (2003) Solution structure of a DNA duplex containing 8-hydroxy-2’-deoxyguanosine opposite deoxyguanosine. J. Mol. Biol. 325, 433-442. (15) McAuley-Hecht, K. E., Leonard, G. A., Gibson, N. J., Thomson, J. B., Watson, W. P., Hunter, W. N., and Brown, T. (1994) Crystal structure of a DNA duplex containing 8-hydroxyguanine-adenine base pairs. Biochemistry, 33, 10266-10270. (16) Choi, Y. J., Chang. S. J., Gibala, K., and Resendiz, M. J. E. (2017) 8-Oxo-7,8-dihydroadenine and 8-Oxo-7,8-dihydroadenosine ― Chemistry, Structure, and Function in RNA; and Presence in Natural Products and in Potential Drug Derivatives. Chem. Eur. J. 23, 6706-6716. (17) Tanaka, M., Jaruga, P., Küpfer, P. A., Leumann, C. J., Dizdaroglu, M., Sonntag, W. E., and Chock, P. B. (2012) RNA oxidation catalyzed by cytochrome c leads to its depurination and crosslinking, which may facilitate cytochrome c release from mitochondria. Free Rad. Biol. Med. 53, 854862. (18) Bischler, T., Hsieh, P-k., Rexch, M., Liu, Q., Tan, H. S., Foley, P. L., Hartlieb, A., Sharma, C. M., and Belasco, J. G. (2017) Identification of the RNA pyrophosphohydrolase RppH of Helicobacter pylori and global analysis of its RNA targets. J. Biol. Chem. 292, 1934-1950. (19) Hayakawa, H., Kuwano, M., and Sekiguchi, M. (2001) Specific binding of 8-oxoguaninecontaining RNA to polynucleotide phosphorylase protein. Biochemistry, 40, 9977-9982. (20) Wu, J., Jiang, Z., Liu, M., Gong, X., Wu, S., Burns, C. M., and Li, Z. (2009) Polynucleotide phosphorylase protects Escherichia coli against oxidative stress. Biochemistry, 48, 2012-2020. (21) Morita, Y., Shibutani, T., Nakanishi, N., Nishikura, K., Iwai, S., and Kuraoka, I. (2013) Human endonuclease V is a ribonuclease specific for inosine-containing RNA. Nat. Commun. 4, 2273. (22) Schmier, B. J., and Shuman, S. (2014) Effects of 3’-OH and 5’-PO4 base mispairs and damaged base lesions on the fidelity of nick sealing by Deinococcus radiodurans RNA ligase. J. Bacteriol. 196, 1704-1712. (23) Simms, C. L., Hudson, B. H., Mosior, J. W., Rangwala, A. S., and Zaher, H. S. (2014) An active role for the ribosome in determining the fate of oxidized mRNA. Cell Reports, 9, 1256-1264. 28

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(44) Chauca-Diaz, A. M., Choi, Y. J., and Resendiz, M. J. E. (2015) Biophysical Properties and Thermal Stability of Oligonucleotides of RNA Containing 7,8-Dihydro-8-hydroxyadenosine. Biopolymers, 103, 167-174. (45) Antonov, I. V., Karpeisky, M. Ya., Padyukova, N. Sh., Yakovlev, G. I., and Sakharovsky, V. G. (1979) On the interaction of pyrimidine specific ribonuclease A with purine nucleotides. Bioorganicheskaia Khimiia, 5, 280-288. (46) Haar, W., Maurer, W., and Rüterjans, H. (1974) Proton-magnetic-resonance studies of complexes of pancreatic ribonuclease A with pyrimidine and purine nucleotides. Eur. J. Biochem. 44, 201-211. (47) Schwarz, F. P. (1988) Interaction of cytidine 3’-monophosphate and uridine 3’-monophosphate with ribonuclease a at the denaturation temperature. Biochemistry, 27, 8429-8436. (48) Li, Y., and Breaker, R. R. (1999) Kinetics of RNA degradation by specific base catalysis of transesterification involving the 2’-hydroxyl group. J. Am. Chem. Soc. 121, 5364-5372. (49) AbouHaidar, M. G., and Ivanov, I. G. (1999) Non-enzymatic RNA hydrolysis promoted by the combined catalytic activity of buffers and magnesium ions. Z. Naturforsch. 54c, 542-548. (50) Lopez-Galindez, C., Lopez, J. A., Melero, J. A., de la Fuente, L., Martinez, C.; Ortiz, J., and Perucho, M. (1988) Analysis of genetic variability and mapping of point mutations in influenza virus by the RNase A mismatch cleavage method. Proc. Natl. Acad. Sci. 85, 3522-3526. (51) Spencer, S. D., Abdul, O., Schulingkamp, R. J., and Raffa, R. B. (2002) Toward the design of ribonuclease (RNase) inhibitors: Ion effects on the thermodynamics of binding of 2’-CMP to RNase A. J. Pharmacol. Exp. Ther. 301, 925-929. (52) Spencer, S. D., and Raffa, R. B. (2004) Isothermal titration calorimetric study of RNase-A kinetics (cCMP→3’-CMP involving end-product inhibition. Pharm. Res. 21, 1642-1647. (53) Schultz, S. J., and Champoux, J. J. (2008) RNase H activity: Structure, specificity, and function in reverse transcription. Virus Res. 134, 86-103. (54) Magner, D., Biala, E., Lisowiec-Wachnicka, J., Kierzek, E., and Kierzek, R. (2015) A tandem oligonucleotide approach for NNP-selective RNA degradation using modified antisense oligonucleotides. PLoS ONE, 10, e0142139. 31

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Scheme 1. 105x47mm (300 x 300 DPI)

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Table 1. Amounts of RNase used per experiment. The text describes Z or 10Z as a dilution factor in the figures. 140x116mm (300 x 300 DPI)

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Figure 1. 20% Denaturing PAGE of RNA 1-4 following treatment with RNase T1 (experiment carried out in triplicate, Fig S1). ONs 5 and 6 also displayed a lack of reactivity towards this RNase (Fig S2). [RNase] – Z = 5, refer to Table 1. 67x128mm (300 x 300 DPI)

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Figure 2. 20% Denaturing PAGE of RNA 1-4 following treatment with RNase A at dilution factors of Z = 6, or 7 (Table 1) Experiments carried out at Z = 2-8 RNase A are included in the supporting materials Fig S3. 121x106mm (300 x 300 DPI)

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Figure 3. 20% Denaturing PAGE of RNA 7-11 following treatment with RNase at varying enzyme concentrations. Z corresponds to the order of magnitude in the dilution factor, e.g., 102 = 100 fold-dilution, see Table 1. 116x99mm (300 x 300 DPI)

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Figure 4. 20% Denaturing PAGE of RNAs 1, 2, 5, and 6 (A); and 14-17 (B); following treatment with RNase A at varying enzyme concentrations. Z corresponds to the dilution factor, e.g., Z = 3 is a 1,000 X dilution (see Table 1 for exact amounts). 163x83mm (300 x 300 DPI)

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Figure 5. Treatment of duplexes of RNA w/wo base pair mismatches G:C, C:C. U:C, and 8-oxoG:C at a single site followed by treatment with RNase A along with recorded Tm values via circular dichroism ([RNA] = 3 µM, 10 mM sodium phosphate – pH 5.5). [RNase] – Z = 6 for 7/8 and Z = 5 for 9/10/11. 142x55mm (300 x 300 DPI)

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Figure 6. Structure of methyl monophosphate derivatives P1-P4 and their corresponding thermograms obtained via ITC. 178x110mm (300 x 300 DPI)

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Figure 7. Treatment of RNA:DNA duplexes after treatment with RNase H displays major and minor cleavages, represented by the size of the arrow. Dotted arrows (green) depict cleavage sites at lower ribonuclease concentrations. Tm values are also shown and were acquired via circular dichroism recorded at 270 nm ([RNA] = 2 µM, RNase H buffer final volume = 7.5 mM KCl, 5 mM Tris-HCl, 0.3 mM MgCl2, 1 mM dithiothreitol [provided by manufacturer]). Electrophoresis was carried out on 20 % denaturing PAGE for all experiments. Z corresponds to the dilution factor, e.g., Z = 2 is a 100 X dilution as detailed in Table 1. (A) Shows reactivity towards RNA:DNA duplexes containing ONs 1, 2, 5, 6 and (B) 7, 10, 12, 13 (Gel shown on Figure S8). 171x220mm (300 x 300 DPI)

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Figure 8. Treatment of RNA:DNA duplexes 21-24 after treatment with RNase H displays major and minor cleavages, represented by the size of the arrow. Tm values are also shown and were acquired via circular dichroism recorded at 270 nm ([RNA] = 2 µM, RNase H buffer final volume = 7.5 mM KCl, 5 mM Tris-HCl, 0.3 mM MgCl2, 1 mM dithiothreitol [provided by manufacturer]). Z corresponds to the dilution factor, e.g., Z = 2 is a 100 X as described in Table 1. Electrophoresis was carried out on 20 % denaturing PAGE for all experiments. 257x138mm (300 x 300 DPI)

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Figure 9. Calculated secondary structure of RNAs 21 and 25 along with their corresponding thermal denaturation transitions, obtained via CD (10 mM Sodium phosphate buffer, pH 5.5). ‘2o struct’ describes formation of a secondary structure, via CD, that is folding of coil into the proposed hairpin. 100x33mm (300 x 300 DPI)

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Figure 10. Known H-bonding interactions involved in the specificity of the enzyme (bottom right) and proposed structures that explain the lack of reactivity of 8-oxoG sites and C8-functionalized guanine rings (bottom left and top right). The image on the bottom right was obtained from the Protein Data Bank (RCSB PDB), as referenced in the text. 153x122mm (300 x 300 DPI)

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Figure 11. Proposed models that explain the reactivity of 8-oxoG and lack thereof in the 8-OMeG, 8-BrG, and 8-oxoA cases. The image on the left was obtained from the Protein Data Bank (RCSB PDB), ref. 63-64; and a pyrimidine ring was drawn to illustrate that space constraints may not be a factor in fitting a purine nucleobase within the binding site; T45 is also highlighted. 260x95mm (300 x 300 DPI)

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