Impact of G-quadruplex RNA oxidation on its conformational dynamics and interaction with ALS-associated TDP-43

Impact of G-quadruplex RNA oxidation on its conformational dynamics and interaction with ALS-associated TDP-43 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Impact of G-quadruplex RNA oxidation on its conformational dynamics and interaction with ALS-associated TDP-43 Akira Ishiguro This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7634186/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Feb, 2026 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the selective degeneration of motor neurons. The primary cause of ALS, whether sporadic or familial, is aging, and recent studies have shown that age-related RNA oxidation plays a role in the early stages of disease onset. This study focused on the vulnerability of G-quadruplex (G4) structures to oxidation and aimed to elucidate the molecular mechanism underlying the conformational changes and their interactions with the binding protein TDP-43. Guanine within G4 structures has a low redox potential, and its substitution with 8-oxoguanine (8OG) can induce structural instability and impair its function as a protein binding signal. In addition, synthetic G4-RNAs modified by oxidation were examined, and results showed that conformational changes are due to different hydrogen bond arrangements, 8OG-A mismatches, and intermolecular G4 formation. The interaction between G4 and TDP-43 decreased in proportion to the substitution rate of 8OG. Furthermore, ALS-associated mutant proteins exhibited reduced binding affinity for oxidized G4s compared with the wild-type. Considering that intra-axonal mRNA transport mediated by G4-binding proteins is essential for the survival and activity of motor neurons, this study will provide important insights into the molecular mechanisms underlying the onset of ALS with aging. Biological sciences/Biochemistry Biological sciences/Biophysics Biological sciences/Neuroscience RNA 8-Oxoguanine (8‐oxoG) TAR DNA‐binding protein 43 (TDP‐43) (TARDBP) amyotrophic lateral sclerosis (ALS) (Lou Gehrig disease) G‐quadruplex aging Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the selective loss of upper and lower motor neurons [ 1 , 2 ]. However, the cause of the selective vulnerability of motor neurons remains unknown, which hinders the development of effective treatments. Approximately 50 ALS-associated and possibly related genes have been identified, most of which encode RNA-binding proteins (RBPs), such as TDP-43 (43-kDa TAR DNA-binding protein, encoded by TARDBP ) and fused in sarcoma (FUS) [ 2 , 3 ]. These RBPs normally contribute to RNA metabolism and intra-axonal mRNA transport via RNA granules formed by liquid–liquid phase separation (LLPS) [ 3 ]. mRNA recognition is highly selective, and guanine quadruplex (G4) is utilized as a common protein- binding signal [ 3 – 7 ]. G4s are noncanonical DNA/RNA structures in which four guanines incorporate alkali metal ions by hydrogen bonding and fold over to stabilize them [ 8 , 9 ]. Therefore, the dysregulation of intra-axonal mRNA transport caused by abnormalities in G4-protein interaction can increase the risk of disease onset [ 3 ]. In support of this hypothesis, ALS-associated TDP-43 and FUS mutant proteins showed reduced binding compared with the wild-type in G4-binding assays [ 5 , 7 ]. In addition, considering cases where imperfections on the G4 side effect the interaction and cause abnormalities in mRNA transport is necessary. Aging is the primary risk factor for ALS, affecting sporadic and familial cases, and the number of patients continues to increase alongside increasing life expectancy [ 10 ]. The age dependence of ALS onset has some possible reasons, with increased oxidative stress being considered as a major risk factor. Oxidative stress arises because of an excess of reactive oxygen species (ROS), such as superoxide radicals, hydrogen peroxide (H 2 O 2 ), and hydroxyl radicals; such ROS are generated as metabolic by products by biological systems [ 11 , 12 ]. Over the last 30 years, the causal relationship between ALS and oxidative stress has been investigated, with most studies targeting oxidative damage to DNA and proteins. However, no ALS-associated gene mutations that are involved in the repair of oxidized DNA and proteins have been reported, and most of the mutations occur in genes that encode for RBPs, axonal transport-related proteins, and redox-active proteins [ 1 , 2 ]. Furthermore, oxidative damage accumulates preferentially in the RNA pool over DNA and proteins [ 13 , 15 , 16 ]. Compared with DNA, which is double stranded, bound to histones, and compartmentalized in the nucleus, RNA has been reported to be 14–25 times more susceptible to oxidation [ 15 ]. In recent years, age-related RNA oxidation has received considerable attention, particularly in relation to neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and ALS [ 17 – 19 ]. Among nucleotides, guanine has the lowest redox potential, and it is easily oxidized. Therefore, G4-RNA composed of guanine nucleotides is highly sensitive to oxidative stress [ 3 , 20 ]. Substituting guanine with an oxidized form, 8-oxoguanine (8OG), leads to the rearrangement of hydrogen bond donors and acceptors on the Hoogsteen ends of the nucleobase, affecting the cation localization and exchange properties [ 21 , 22 ]. The accumulation of 8OG has been reported in aged neurons, ALS patients, and animal models [ 23 , 24 ]. Of the ALS-associated and possibly related genes identified, 19 code for RBPs and RNAs [ 3 ]. All of these genes encode G4-binding proteins (G4BPs), G4-regulatory proteins, or abundant G4-forming sequences, raising concerns about a link between ALS and guanine oxidation [ 3 ]. Although the destabilization of G4s by oxidative modifications has been primarily studied as a mechanism for the switch-on of oxidative stress–responsive G4-containing promoters and mRNAs [ 21 , 25 – 27 ], whether the loss of conformation affects interactions with G4BPs has not been demonstrated. RNA oxidation does not result from neuronal cell death, but an early event associated with pathogenic mechanisms [ 28 , 29 ]. Consequently, redefining this dysregulated process is essential. In this study, G4-RNAs containing 8OG substitutions were used, which mimic age-related oxidative damage, to analyze its comprehensive conformational changes and interactions with TDP-43, a G4BP with high binding specificity. This study, which focuses on the relationship between G4-RNA oxidation and ALS-associated G4BP, will provide novel insights into the molecular mechanism of age-related ALS onset. Results G4 conformation is affected by oxidative modifications First, immunocytochemistry was performed using the human neuroblastoma cell line GOTO to visualize the effect of oxidative stress on G4-RNA/DNAs in cells (Fig. 1 A). The cells were treated with hydrogen peroxide for 30 min [ 28 ]. Then, the medium was exchanged, and the cells were fixed 12 h later and probed with the anti-G4 antibody BG4 (Fig. 1 B). Fluorescence images showed that cells treated with hydrogen peroxide had reduced the G4 signals compared with untreated controls. However, this result does not necessarily lead to the conclusion that oxidative modification of G4 caused the conformational change. G4BPs bind to entire G4, making them difficult to detect adequately with antibodies that have conformational binding properties [ 6 , 30 ]. Fay et al. proposed the classification of G4BPs into those that bind and prevent the structure (Group 1; FUS; α-synuclein; zinc finger protein 106, Zfp106 etc.) and those that stabilize the structure (Group 2; TDP-43; Fig. 1 C) [ 30 – 32 ]. Both groups can exhibit protection from recognition by anti-G4 antibodies. In support of this hypothesis, the fluorescence images showed that the G4 and TDP-43 signals barely overlapped (Fig. 1 B, Supplementary Fig. S1 ). In cells, the number of foci detected by anti-G4 antibodies is orders of magnitude lower than the number of G4-forming sequences predicted genome wide [ 9 ]. Hence, total RNAs from stressed cells were purified and probed using the G4-specific fluorophore, Thiazole orange biotin (TO1B) to quantitatively evaluate the effect of oxidative modifications on G4-RNA [ 33 ]. As a result, the fluorescence intensity of TO1B decreased in a hydrogen peroxide concentration-dependent manner (Fig. 1 D). This result is due to conformational alterations of G4-RNAs, excluding the effect of protein binding. Oxidative treatments in these cell-based assays may also affect transcription and RNA stability. Thus, precisely quantifying the effect of oxidative RNA modifications on the direct interaction of TDP-43 with G4s is necessary. Therefore, synthetic G4-RNAs derived from two types of mRNA 3′-UTR, namely, PSD-95 (postsynaptic density protein 95) and CaMKIIα (calcium/calmodulin-dependent protein kinase type II subunit alpha), were prepared [ 3 – 7 , 33 ] (Fig. 2 A and B). Based on previous reports, these two mRNAs are transported to neurites in a G4-dependent manner and translated locally [ 4 , 34 ]. These G4-RNAs were synthesized using phosphoramidites containing guanine mixed with 10%, 20%, or 40% 8OG to serve as oxidized model G4-RNAs (Supplementary Table S1 ). The 8OG dose-dependent comprehensive modification of these synthetic model RNA mixtures was demonstrated by Western blotting using an anti-8OG antibody and electrophoresis (Fig. 2 C and D, Supplementary Fig. S2 and Fig. S3). Electrophoresis was performed under nondenaturing conditions, and staining with an intercalating fluorescent agent confirmed that the signal was remarkably reduced depending on the ratio of 8OG (Fig. 2 D, and Supplementary Fig. S3). A slow mobility was observed for the most highly oxidized CaMKIIα-G4 (40% 8OG). The difference in staining levels may be due to a conformational change in G4, which inhibits the binding of the intercalating agent. 8OG destabilizes the structure by preventing the formation of Hoogsteen hydrogen bonds [ 20 , 21 , 34 ] while it has been reported to increase the stability [ 26 , 36 ]. Conformational alterations caused by guanine oxidation In evaluating the conformational effects of model G4-RNAs with 8OG substitutions, Circular dichroism (CD) spectroscopy measurements were performed. RNA forms only parallel stranded G4s, and the CD spectrum shows a negative peak at around 240 nm and a positive peak at around 265 nm, which are a characteristic of the structure. However, the CD spectra of G4-RNAs derived from PSD-95 and CaMKIIα mRNAs showed different effects of the substitution with 8OG (Fig. 3 A and B). CaMKIIα-G4 showed a decrease in positive and negative peaks in an 8OG dose-dependent manner. This result indicates that oxidative modifications of CaMKIIα-G4 structures often contribute to destabilization. In contrast, the spectrum of PSD-95-G4 was reduced at 40% 8OG, whereas both peaks were enhanced in RNA containing 10% and 20% 8OG, with the positive peak shifted slightly to the longer wavelength side (Fig. 2 A). The comparison of the CD spectra of PSD-95-G4 and RNAs with 8OG substitutions leads to two possibilities: (1) Substitution with 8OG stabilized the structure, or (2) the presence of a poly-A configuration led to an 8OG-adenine mismatch pair. Adenine frequently pairs with the Hoogsteen edge of 8OG, causing structural alteration [ 27 ]. The associated RNA regions that could be formed by the 8OG-A mismatch may enhance the spectrum close to the positive peak of G4. In investigating these possibilities, a fluorescent turn-on assay was performed using TO1B (Fig. 3 C and D). The measurements showed that the fluorescence intensity of not only CaMKIIα-G4 but also PSD-95-G4 decreased in response to the rate of 8OG substitution. The decrease in TO1B signals in the 10% 8OG and 20% 8OG model RNA mixtures of PSD-95 indicated instability as a G4 structure, which might be due to an 8OG-A mismatch. The slight shift of the peak to longer wavelengths could be due to this effect [ 40 , 41 ]. Hence, in this study, detection was performed using naphthyridine–azaquinolone (N-A), which binds to the G-A mismatch [ 42 ]. N-A exhibits a UV absorption peak around 320 nm, which decreases upon binding to the G-A mismatch (Fig. 3 E). The absorbance at 320 nm did not change for nonoxidized G4, but it decreased for all three types of oxidized G4s (Fig. 3 F). Under oxidative stress conditions, 8OG-A mismatch is observed in double-stranded DNA/RNA and codon–anticodon interactions [ 27 ], which indicated that this phenomenon also occurs in G4-RNA. In addition, the decrease in the 320-nm peak caused by mismatch formation was more pronounced for the 10% substituted RNA than for the 20% and 40% substituted RNAs. The positive peak in the CD spectrum was also highest at 10% 8OG, indicating that a transient G4 conformation favors mismatch formation. Thus, mismatch formation does not depend on the substitution rate of 8OG but is further promoted by the stable proximity of adenine to 8OG through transient G4 formation (Fig. 3 G). In addition, highly oxidized RNAs strongly inhibit the formation of the G4 structure, thereby reducing the accessibility of 8OG to A and suppressing mismatch formation. Furthermore, CaMKIIα-G4, which has a thermodynamically stable structure without an A-loop [ 6 ], is more sensitive to 8OG substitution, that is, changes in hydrogen bond positions, compared with PSD-95-G4 (Fig. 3 C and D). These results imply that there are different types of G4 conformational changes upon guanine oxidation (Fig. 3 G), and the combination of CD spectroscopy with other analytical techniques could avoid misinterpretations. G4 oxidation reduces TDP-43 binding caused by conformational alterations To date, no analysis has been performed on the interaction between G4BP and G4 structures altered by 8OG substitution. Most G4BPs contain intrinsically disordered regions, making them difficult to purify and handle, and few proteins have been demonstrated to bind exclusively to G4s [ 6 , 43 ]. Previously, our group successfully purified full-length, tag-free TDP-43 and FUS, demonstrated their interactions with DNA/RNAs, and revealed differences in their binding specificity [ 5 , 6 ]. FUS binds to all three types of G4s (parallel, hybrid, and antiparallel) and a hairpin structure, whereas TDP-43 recognizes only parallel G4 and does not bind to a hybrid, antiparallel, and pUG fold [ 6 , 44 ] (Supplementary Fig. S4, Fig. S5, and Fig. S6). Therefore, TDP-43, which has high binding specificity, is suitable for measuring the interactions related to the G4 conformational alterations in this study. The extent to which the 8OG substitution affects the interaction through changes in the G4 structure was confirmed by a gel shift assay. The binding of TDP-43 to fluorescently labeled nonoxidized G4 was quantified by competition with unlabeled RNA containing 0, 10%, 20%, or 40% 8OG substitutions. These nonlabelled competitor RNAs were added at threefold and 10-fold the fluorescently labeled nonoxidized G4 probe level. The inhibitory effect of PSD-95-G4 decreased as the proportion of 8OG increased, and it was almost undetectable at 40% (Fig. 4 A and Supplementary Fig. S7). On the contrary, CaMKIIα-G4 was more sensitive to 8OG, and it showed no inhibitory effect even at 10% substitution (Fig. 4 B and Supplementary Fig. S7), which was consistent with the results of the CD spectra and TO1B turn-on assay (Fig. 3 ). These results indicated that the oxidation of G4 induces conformational alterations that reduce the interaction with TDP-43. In addition, the effects of 8OG are not identical, but they vary greatly depending on the type of G4. PSD-95-G4 has a higher redundancy possibly because of the flexible A-loop, and it is more resistant to oxidation. By contrast, the dense and thermodynamically stable CaMKIIα-G4 is less redundant and is disruptively altered by the 8OG substitution. However, CaMKIIα-G4, which contains the most 8OG (40% 8OG), showed slight binding inhibition (Fig. 4 B and Supplementary Fig. S7). Our group has previously demonstrated that TDP-43 stabilizes the G4 structure as group 2 G4BP [ 6 ]. Therefore, in this study, CD spectroscopy was used to examine whether these conformational changes occurred upon the binding of oxidized PSD-95-G4s (10% and 20% 8OG) with TDP-43 (Fig. 4 C). Upon binding to TDP-43, the structure of the nonoxidized G4 was barely altered [ 6 ], but the positive and negative peaks decreased for both oxidized RNAs. Therefore, the conformational alternation caused by the 8OG-A mismatch was suppressed by binding with TDP-43. Considering that TDP-43 cannot bind to RNA with a disrupted G4 structure, it should not repair the mismatch or rearrangement of the hydrogen bond. It likely binds to the transition intermediate G4 and prevents the formation of an 8OG-A mismatch (Fig. 4 D). Guanine oxidation induces intermolecular G4 formation The most highly oxidized CaMKIIα-G4 (40% 8OG) slightly but significantly inhibited protein binding. (Fig. 4 B). In addition, this RNA was observed to exhibit retarded mobility upon electrophoresis under nondenaturing conditions (Fig. 2 D). It was hypothesized that molecules that cannot form intramolecular G4s might adopt intermolecular structures. In examining this hypothesis, asymmetric intermolecular G4 formation was attempted using the short G-rich RNA G 3 UG 3 (Fig. 5 A). This short RNA has only one loop-forming uracil, and symmetric intermolecular G4 structures are relatively unstable, and it also tends to form asymmetric intermolecular G4 structures (Fig. 5 B) [ 45 , 46 ]. SYBR Green II staining showed an increase in CaMKIIα-G4 (40% 8OG) signal upon the addition of G 3 UG 3 , indicating the formation of asymmetric intermolecular G4 structures (Fig. 5 , C and D, and Supplementary Fig. S8). No synergistic effect was observed with the addition of G 3 UG 3 in PSD-95-G4 (40% 8OG). When lithium, which does not allow stable G4 formation, was used instead of potassium, no changes were observed upon the addition of G 3 UG 3 . In both RNAs with 20% 8OG substitutions, no increase in the signal caused by G 3 UG 3 could be detected (Supplementary Fig. S9 and Fig. S10). Therefore, the inhibition of protein binding by oxidized CaMKIIα-G4 (40% 8OG; Fig. 4 B) was due to the promotion of intermolecular G4 formation. In this study, the formation of intermolecular G4s is likely detrimental to the regulation of mRNA transport. Consecutive guanine sequences are common in mRNAs, tRNAs, rRNAs, and ncRNAs. The abnormal intermolecular G4 formation with other G-rich RNAs may not only inhibit normal G4-mRNA transport but also lead to undesirable RNA metabolism and dysregulation. G4 oxidation further increases the risk of ALS-associated TDP-43 mutations Our group previously investigated the effects of 10 ALS-associated amino acid substitution mutations in TDP-43 using surface plasmon resonance (SPR) analysis, demonstrating that all these mutations exhibit reduced interaction with G4-RNAs [ 5 ]. TDP-43 interacts with G4s through three modules: two RNA recognition motifs (RRMs) with high affinity for RNA and a glycine-rich domain that modulates specific binding (Fig. 6 A). Most disease-associated mutations are located within the disordered glycine-rich region, indicating a link between binding specificity and disease onset [ 33 , 47 , 48 ]. The structural changes induced by G4 oxidation could pose a greater risk to the TDP-43 variants associated with ALS. To explore this finding, the 10 ALS-associated mutant proteins were used to analyze the effect of G4 oxidation on their interactions, using gel shift assays with a PSD-95-G4 probe and oxidized RNA competitor (Fig. 6 B). The gel shift assays confirmed that all mutant proteins exhibited reduced binding to G4, which is consistent with previous SPR results [ 5 ]. However, differences emerged in their inhibitory effects when competed with nonfluorescently labeled oxidized G4 (10%) compared with the wild-type (Fig. 6 C and D, and Supplementary Fig. S11). Notably, all 10 mutants were less affected by unlabeled oxidized RNA than the wild-type (Fig. 6 E), indicating a weaker affinity for oxidized RNA. Among these, the G287S and P363A mutations, which showed the most significant difference from the wild-type, are located at the center of two peaks in the confidence values within the disordered regions (Fig. 6 A). This phenomenon likely results from the combined effects of amino acid substitutions and conformational alterations of G4 within regions normally stabilized by G4 interactions. Overall, these findings indicate that the decreased interaction between ALS-associated TDP-43 mutants and G4-RNA is further exacerbated by guanine oxidation. However, at present, data regarding the earlier onset or shorter disease duration in patients with G287S and P363A mutations are limited. As more genotypic and phenotypic data are collected from patients, the causal relationship between TARDBP mutations and age-related G4 oxidation will become more evident. Discussion In recent years, oxidative RNA damage has gradually come to the forefront of attention in the areas of cancer, metabolic diseases, cardiovascular diseases, and neurodegenerative diseases [ 17 , 29 ]. A variety of evidence, including studies of human and animal models, indicates that RNA oxidation is a hallmark of neurons in the aging brain and is more pronounced in neurons at the early stages of age-related neurodegenerative diseases [ 29 ]. Guanine has the lowest redox potential among nucleobases, and it is readily oxidized, leading to the formation of oxidized RNAs either through the direct oxidation of polynucleotide bases or the incorporation of oxidized bases during normal RNA synthesis by RNA polymerases [ 48 ]. Thus, G4s formed from four consecutive G regions were susceptible to oxidation, and the resulting conformational abnormality manifested as a reduced signal in cultured cells stained with an anti-G4 antibody (Fig. 1 ). However, although the conformational defects of G4-RNA caused by oxidation within cells could be quantified using extracted and purified RNA, detecting the interaction with binding proteins in cells was difficult. In enabling precise comparison and quantification, RNA mixtures were used in which guanines were randomly substituted with 8OG to mimic oxidation in cells, and the conformational changes of G4s caused by oxidative modifications, as well as their interactions with TDP-43, were analyzed. The effect of G4 oxidation varies depending on the sequence and loop configuration but comprehensively leads to a shift toward a less stable state, as quantitatively demonstrated by CD spectroscopy, and TO1B turn-on assays (Fig. 2 E and Fig. 3 ). One of the unexpected effects of oxidative modification on the G4 structure was the formation of an 8OG-A mismatch within the A-loop of PSD-95-G4 (Fig. 3 ). The mismatch formation was most efficient in RNA with 10% 8OG substitution rather than 20% or 40%, indicating that the proximity caused by G4 assembly is important to the formation. The decrease in the absorbance of mismatch-bound N-A and the inhibition of mismatch formation in the presence of TDP-43 also supported these predictions (Fig. 3 F and Fig. 4 C). Another unexpected effect was that the oxidation of guanine resulted in the formation of an intermolecular G4. Considering that misalignment of hydrogen bonds is fatal to the formation of intramolecular G4s, the remaining nonoxidized consecutive guanines form the intermolecular conformation. These guanines may even form abnormal G4s not only with various RNAs containing consecutive guanine sequences but also with DNA. Finally, the structural abnormalities caused by G4 oxidation led to reduced binding to TDP-43, and all 10 ALS-associated TDP-43 mutant proteins showed a reduced ability to bind to oxidized G4s compared with the wild-type (Fig. 6 ). These results imply that even low levels of oxidative damage to G4s can lead to dysregulation through synergistic effects with protein mutations. Recently, a growing number of studies have reported that G4-RNAs are enriched within transient biomolecular condensates called stress granules, which are formed by LLPS as a cellular response to heat stress, endoplasmic reticulum stress, and oxidative stress, etc. [ 49 – 51 ]. The enrichment of G4-RNAs and RBPs in stress granules is consistent with the aims of this study and is considered as an ideal molecular mechanism for protecting RNAs. Under short-term oxidative stress, RNA oxidation does not occur immediately and appears to be protected [ 28 ]. Furthermore, G3BP1 (Ras GTPase-activating protein binding protein 1) contributes to stress granule assembly, and its knockdown reduces stress granule size and impairs the preservation of mRNAs [ 52 , 53 ]. The dysfunction of G3BP1 could be involved in the onset of neurodegenerative diseases, including ALS, and G3BP1 is also a G4BP [ 54 , 55 ]. Thus, a system that preferentially enriches G4-mRNAs should be particularly neuroprotective, and dysregulation could pose risks to motor neurons, where the axonal transport of G4-mRNA is essential. To date, most studies on cellular damage caused by RNA oxidation have focused on the translation machinery. Oxidized RNAs cause miscoding and stalled ribosomes, leading to the production of abortive or defective peptides/proteins [ 27 ]. 8OG-containing mRNA has been shown to reduce protein production and fidelity in cell-free translation systems and cultured cells [ 56 , 57 ]. Nonetheless, the findings indicate that the selective vulnerability of motor neurons to RNA oxidation in ALS should not be considered solely in terms of its effects on the translation machinery. Most of the genes causative for ALS encode RBPs, such as TDP-43, FUS, Senataxin (SETX), Zfp106, Angiogenin (ANG), Ataxin-2 (ATXN2), heterogeneous nuclear ribonucleoprotein RNA A1 (hnRNPA1), heterogeneous nuclear ribonucleoprotein A3 (hnRNPA3), hnRNPA2/B1 (heterogeneous nuclear ribonucleoprotein A2/B1), ewing sarcoma RNA-binding protein (EWSR1/EWS), T cell intracellular antigen-1 TIA1, RNA-binding motif protein 14 (RBM14), TATA-binding protein associated factor 15 (TAF15), and splicing factor proline and glutamine rich (SFPQ), all of which either directly bind to G4 or regulate G4-forming sequences [ 32 ]. In addition, the intronic hexanucleotide repeat expansion (GGGGCC)n in C9ORF72 (chromosome 9 open reading frame 72) that forms numerous G4 structures is the most common genetic cause of ALS [ 58 ]. The effects of G4 oxidation confirmed in this study likely extend to all these factors, which could increase the risk of developing ALS. G4-dependent long-distance mRNA transport is more important in motor neurons than in other cells [ 4 , 34 ]. Therefore, motor neurons suffer most from the dysregulation of G4-dependent long-distance mRNA transport. Collectively, the vulnerability of G4s to oxidation leads to diverse conformational alternations and reduced binding to proteins involved in axonal mRNA transport. This effect may be most pronounced in neurons, indicating that G4-RNA oxidation is a molecular hallmark of aging that contributes to increased ALS risk. Materials and methods Cell culture The GOTO human neuroblastoma cell line (Japanese Collection of Research Bioresources, JCRB0612) was cultured in RPMI 1640 medium (Thermo Fisher Scientific Inc. Waltham, Massachusetts, USA) supplemented with 10% fetal calf serum and 1% penicillin–streptomycin (Thermo Fisher Scientific Inc. Waltham, Massachusetts, USA) at 37°C in a 5% CO 2 atmosphere. Immunofluorescence staining Immunofluorescence staining of cultured cells was performed as previously described 4 , using a rabbit polyclonal anti-TDP-43 antibody (Cell Signaling Technology Inc., #3449 Danvers, Massachusetts, USA) and the mouse monoclonal anti-G-quadruplex antibody BG4 (Absolute Antibody Ltd., Ab00174-1.1 Redcar, UK). Thiazole orange biotin (TO1B) turn-on assay Total cellular RNAs were extracted using the TRIzol reagent (Thermo Fisher Scientific Inc. Waltham, Massachusetts, USA) in accordance with the manufacturer's instructions. Cellular RNA (20 µg) or synthetic RNA (20 pmol) was dissolved in 180 µL of buffer (20 mM PIPES-KOH, pH 6.8, 0.1 mM MgCl 2 , and 150 mM KCl), then mixed with 20 µL of 1 µM thiazole orange-3PEG biotin (TO1B; Applied Biological Materials Inc. Richmond, British Columbia, Canada), dissolved in the same buffer, and incubated at 25°C for 5 min. Afterward, ligand-enhanced fluorescence spectra were measured using a fluorescence spectrometer (Shimadzu RF-5300PC, Shimadzu Corporation) with an excitation wavelength of 505 nm and emission spectra ranging from 520 to 600 nm [ 4 ]. RNA/DNA The synthesized oligonucleotides, which were used in this study, were obtained from GeneDesign, Inc., and FASMAC Co., Ltd. (Table S1 ). RNA staining In staining dot-blotted RNAs on the membrane, the membrane was immersed in a 0.05% toluidine blue solution (Fujifilm Co., Ltd.) for 20 s and then washed with water. RNA separated by electrophoresis on an agarose gel under native conditions was stained with SYBR Green II (Takara Bio Inc.) in accordance with the manufacturer's instructions and visualized using the Typhoon 9410 imaging system (GE Healthcare). Immunoblotting RNAs (2.5 pmol) were blotted onto a nylon membrane (Roche Co., Ltd. Basel, Switzerland) and cross-linked using an Ultraviolet (UV) cross-linker (Funakoshi Co., Ltd. Tokyo, Japan). Immunoblotting was performed using an anti-8-oxo-guanine (8-oxoG) mouse monoclonal antibody (Merck Biopharma Co., Ltd.; SAB5200010 Tokyo, Japan) and an anti-mouse IgG-HRP antibody (Jackson Immuno Research Laboratories, Inc.; 715-035-151 West Grove, Pennsylvania, USA) to confirm guanine oxidation. Furthermore, signals were visualized and quantified using the Typhoon 9410 imaging system. Circular dichroism (CD) spectral analysis The estimated G4 structures of the synthesized RNAs were confirmed by CD spectral analysis using a 2 µM oligonucleotide in a buffer containing 50 mM PIPES (sodium 2-[4-(2-sulfoethyl)piperazin-1-yl] ethanesulfonate)–KOH (pH 6.8), 150 mM KCl, and 1 mM MgCl 2 at 25°C, as previously described [ 4 ], with a spectropolarimeter (JASCO J-820). Before measurement, each RNA sample was heated at 98°C for 5 min and then allowed to cool to room temperature. The scans were repeated five times, and Jasco Spectro Manager software was used to compile the average spectra. The x-axis represents the wavelength, while the y-axis indicates the molar CD (Δε). For binding experiments with the TDP-43 protein, an additional final concentration of 1.25% glycerol, prepared from the protein stock solution, was included. UV absorption measurement of naphthyridine–azaquinolone (N-A) hybrids Before use, each oxidized RNA sample (6 µM) prepared in a buffer containing 20 mM PIPES-KOH (pH 6.8), 150 mM KCl, and 1 mM MgCl 2 (40 µL) was heated at 98°C for 3 min and then cooled to room temperature. Subsequently, 10 µL of the 100-µM N-A solution (Synthonix Inc.; AC80704) prepared in buffer containing 0.1% (w/v) dimethyl sulfoxide was added to the sample. After incubation for 15 min at room temperature, absorption was measured using a spectrophotometer (Hitachi High-Tech Co., Ltd.; U-2800). TDP-43 Purified, no-tag, full-length dimeric forms of recombinant human TDP-43 (wild-type and mutant proteins) derived from Escherichia coli were prepared as previously described [ 4 , 5 ]. The protein storage buffer contained 10% glycerol, 20 mM PIPES-KOH (pH 6.8), and 150 mM KCl. The wild-type protein was confirmed to exhibit the same binding activity and specificity for G4 structures as the protein was immunopurified from human cultured cells [ 4 ]. Gel shift assay The gel shift assay was performed as previously described [ 33 ]. Fluorescently labeled oligonucleotides at the 5′-end, including Ax647 PSD-95-G4 and Ax647 CaMKIIα-G4 (0.5 pmol each), were incubated with TDP-43 (2 pmol) in a 10-µL reaction buffer (containing 10% glycerol, 20 mM PIPES–NaOH, pH 6.8, 1 mM MgCl₂, and 150 mM KCl) in the absence or presence of competitor RNAs. The buffer also contained 0.1 mg/mL bovine serum albumin and 0.05 mg/mL poly-dI-dC (Thermo Fisher Scientific Inc.). Incubation was performed at 25°C for 30 min. Then, the samples were loaded onto a 0.5× TBE (Tris-Borate-EDTA; pH 6.8) buffer containing 1% agarose gel and electrophoresed at 50 V for 60 min at 25°C. The gels were visualized and quantified using the Typhoon 9410 imaging system, as previously described [ 33 ]. Intermolecular G4 formation The oxidized model G4-RNAs (20 pmol) were mixed with the absence or presence of G 3 UG 3 (20 pmol) in a 10-µL buffer containing 20% glycerol, 20 mM PIPES–NaOH (pH 6.8), 1 mM MgCl₂, and 150 mM KCl or 150 mM LiCl. The samples were heated at 98°C for 5 min and then cooled to room temperature. Each sample was loaded onto a 1% agarose gel containing 0.5 × TBE and electrophoresed at 50 V for 60 min at 25°C. The gels were stained with SYBR Green II and analyzed using Molecular Imager FX (Bio-Rad Laboratories, Inc. Hercules, California, USA). Declarations Author contributions statement Conceptualization, investigation, data curation, visualization and writing: AI Competing interest The author declares no competing interests. Supporting information This article contains supporting information. Funding This work was supported by the Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research (22K07032, 23H04265 and 25K10307) and SERIKA FUND to AI. Author Contribution Conceptualization, investigation, data curation, visualization and writing: AI Acknowledgement I thank Dr. M. Ishiguro for support and helpful advice. Data Availability The data sets generated during and/or analyzed during the current study are available from the corresponding authors on reasonable request. ( [email protected] ). References Boillée, S., Velde, V., Cleveland, D. W. & C. & ALS: a disease of motor neurons and their nonneuronal neighbors. Neuron 52 , 39–59 (2006). Taylor, J. P., Brown, R. H. Jr & Cleveland, D. W. 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06:19:10","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4842953,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineAIFig6.png","url":"https://assets-eu.researchsquare.com/files/rs-7634186/v1/25e55eaf56efd278d39aa85b.png"},{"id":93744432,"identity":"e7104cd0-7488-41fe-96ee-afccd6a44f3b","added_by":"auto","created_at":"2025-10-17 06:11:11","extension":"xml","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":127095,"visible":true,"origin":"","legend":"","description":"","filename":"f5756055eb0c469fba5c1215e60621911structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7634186/v1/f6e46d42cf8c00f2eb922512.xml"},{"id":93744427,"identity":"03ddeac6-92c8-41a9-826e-d95525dbd7e1","added_by":"auto","created_at":"2025-10-17 06:11:10","extension":"html","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":138641,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7634186/v1/6f0c1e35e8367c83e3191a73.html"},{"id":93744413,"identity":"5c0a133e-3c73-457c-8744-50259ab60222","added_by":"auto","created_at":"2025-10-17 06:11:09","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":11318712,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOxidative stress induced the conformational change of G4s.\u003c/strong\u003e (A) Flowchart of the cellular oxidative stress experiment. Twelve hours after the addition of hydrogen peroxide, the GOTO neuroblastoma cells were fixed and immunostained. Then, total RNA was purified from the cells and used for the TO1B turn-on assay. (B) Representative images showing the localization of the TDP-43 and G4 structures, which were visualized by immunofluorescence staining using anti-TDP-43 and anti-G4 antibodies. On the right, differential interference contrast images are overlaid with nuclear staining using DAPI (4′,6-diamidino-2-phenylindole) in white. (C) Binding model of G4BPs. The proteins are classified into two groups: those that bind to prevent G4 formation (group 1) and those that stabilize G4 structures (group 2). (D) Measurement of G4 levels using the G4-specific fluorescent sensor TO1B. The fluorescence emission spectra of TO1B were measured after adding RNA extracted from untreated cells and cells oxidatively stressed with 20, 40, and 80 μM hydrogen peroxide. The G4-independent fluorescence intensity is also shown as a control. The graph on the right displays the average fluorescence intensity at 530 nm. The experiments were performed in triplicate, and the values on the y-axis represent the mean ± SEM. Statistical significance was determined using a two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"AIFig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7634186/v1/2f4e540e4be36c35d937e02a.jpg"},{"id":93744434,"identity":"d89395a0-8512-4537-9882-02b9f384618e","added_by":"auto","created_at":"2025-10-17 06:11:11","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":10355403,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOxidized model G4-RNAs used in this study. \u003c/strong\u003e(A, B) G4-forming sequences of the PSD-95 and CaMKIIα mRNA 3′ UTRs. The G4-forming nucleotides (underlined) were predicted using the web-based servers QGRS Mapper [59] and RNAfold [60]. The predicted G-quadruplex structures by RNAfold, along with their respective minimum free energies, are also displayed. The accuracy of these G4 structural predictions was previously confirmed by hydroxyl radical RNA footprinting [7]. (C) Dot–blot Western blot analysis of RNAs. In addition, RNAs (10 pmol) with 8OG substitution rates of 0%, 10%, 20%, and 40% were blotted onto a nylon membrane and detected using an anti-8OG antibody. The same membrane stained with toluidine blue is also shown. (D) Agarose gel electrophoresis. The nonoxidized and oxidized forms of three types of RNAs (25 pmol each), namely, PSD-95-G4 and CaMKIIα-G4, were electrophoresed on a 1% agarose gel containing 0.5× TBE under native conditions. The gel was stained with the intercalating fluorescent dye SYBR Green II. The red arrows indicate the positions of the bands, whereas the blue arrow indicates an overshifted band.\u003c/p\u003e","description":"","filename":"AIFig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7634186/v1/b7edddb1d055949f5531cfeb.jpg"},{"id":93744404,"identity":"17599ca6-87bd-48ca-9270-b0301238e4ed","added_by":"auto","created_at":"2025-10-17 06:11:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":370709,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConformational alterations caused by guanine oxidation.\u003c/strong\u003e (A, B) PSD-95-G4 and CaMKIIα-G4, as well as their respective 8OG-substituted RNAs (substitution rates of 10%, 20%, and 40%). The spectra were measured five times, and the mean values are shown. (C, D) Quantification of G4 levels using TO1B. Fluorescence emission spectra were recorded for two G4-RNAs (PSD-95 and CaMKIIα) and their 8OG-substituted counterparts (10%, 20%, and 40%) upon binding to TO1B. G4-independent fluorescence signals are also displayed. The right graph shows the average fluorescence intensity at 530 nm. All experiments were performed in triplicate, and the data are presented as mean ± SEM. Statistical significance was assessed using a two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. (E) Chemical structure of the naphthyridine–azaquinolone hybrids (N–A). (F) UV absorption measurement of N-A. In the absence of RNA, N-A exhibits an absorption maximum at 320 nm, as previously reported [41]. As the G-A mismatch increases, the intensity of these absorption peaks decreases, and the peaks undergo a red shift from 320 nm. The graph on the right shows the mean absorption at 320 nm. The experiments were performed in triplicate, and the values on the y-axis represent the mean ± SEM. Statistical significance was determined using a two-tailed Student's \u003cem\u003et\u003c/em\u003e-test. (G) Schematic diagram of the two types of conformational defects caused by G4 oxidation. Type 1 is a well-known G4 conformational alteration resulting from a different arrangement of hydrogen bonds caused by 8OG. Type 2 is the structural change that leads to the formation of the G-A mismatch observed in this study. In this case, the formation of G4 as a transition intermediate is important to the development of the 8OG-A mismatch.\u003c/p\u003e","description":"","filename":"AIFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7634186/v1/491db7bea115698cad9e6a12.png"},{"id":93744409,"identity":"856bc8ee-6016-4e7c-b334-97f9ed510bff","added_by":"auto","created_at":"2025-10-17 06:11:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":258734,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eG4 oxidation reduces its interaction with TDP-43. \u003c/strong\u003e(A, B) Agarose gel mobility shift assay. Fifty nanograms (0.5 pmol) of fluorescently labeled G4 probes (\u003csub\u003eAx647\u003c/sub\u003ePSD-95-G4 or \u003csub\u003eAx647\u003c/sub\u003eCaMKIIα-G4) was mixed with two pmol of TDP-43 and the indicated unlabeled RNA competitors (threefold or 10-fold molar excess relative to the probe) and then electrophoresed under nondenaturing conditions. The shifted bands were quantified, and the mean and standard error of the mean (± SEM) were calculated from three independent experiments and displayed in the graph. The y-axis represents the mean ± SEM. Statistical significance was determined by two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. The red arrow indicates unexpected inhibition by the oxidized form of CaMKIIα-G4 (40% 8OG). These smear signals, indicated by dagger (†), have been confirmed to be the result of dissociation during electrophoresis [32]. (C) CD spectra of PSD-95-G4 and oxidized RNAs (2 μM) in the absence and presence of TDP-43 (2 μM). The scans were repeated five times, and the protein spectra were subtracted; the mean values are shown. The CD spectrum of TDP-43 without RNA is also included. (D) Schematic illustration of G4 stabilization by TDP-43. The CD spectra of the oxidized RNA showed a decrease in the positive peak in the presence of TDP-43, indicating the stabilization of the transition intermediate G4 conformation.\u003c/p\u003e","description":"","filename":"AIFig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7634186/v1/fede2a15a2f31955b2bbad64.png"},{"id":93744412,"identity":"2cc00dcc-a072-44a2-b6ad-64059f5fdbc4","added_by":"auto","created_at":"2025-10-17 06:11:09","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":11502818,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntermolecular G4 formation.\u003c/strong\u003e (A) A short G-rich probe, G\u003csub\u003e3\u003c/sub\u003eUG\u003csub\u003e3\u003c/sub\u003e, and schematic diagram of the G4 conformation. (B) CD spectra of G\u003csub\u003e3\u003c/sub\u003eUG\u003csub\u003e3\u003c/sub\u003e measured in the presence or absence (control) of 150 mM KCl. The scans were repeated five times, and the compiled mean values are shown.\u003cem\u003e \u003c/em\u003e(C) The indicated 40% oxidized RNAs (20 pmol) were mixed with the absence or presence of G\u003csub\u003e3\u003c/sub\u003eUG\u003csub\u003e3\u003c/sub\u003e (20 pmol) and electrophoresed on 1% agarose gel under native conditions. The gels were stained with the intercalating fluorescent dye SYBR Green II, and the individual signals were quantified. The graph below shows the fold change after the addition of G\u003csub\u003e3\u003c/sub\u003eUG\u003csub\u003e3\u003c/sub\u003e, which was calculated by subtracting the signal for G\u003csub\u003e3\u003c/sub\u003eUG\u003csub\u003e3\u003c/sub\u003e alone from the value for mixing 40% oxidized PSD-95 or CaMKIIα RNAs with G\u003csub\u003e3\u003c/sub\u003eUG\u003csub\u003e3\u003c/sub\u003e. The experiments were performed in triplicate, and the data are presented as the mean ± SEM. (D) Schematic diagram of the intermolecular G4 formation. Molecules that cannot form intramolecular G4 structures adopt intermolecular conformations. These structures can exhibit symmetric bimolecular conformations or form G4s asymmetrically with other G-rich sequences.\u003c/p\u003e","description":"","filename":"AIFig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7634186/v1/007f5a4f2181e465d9b0d6c7.jpg"},{"id":93744421,"identity":"f35e66c9-8f7b-4f41-9518-53265b21a04e","added_by":"auto","created_at":"2025-10-17 06:11:10","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":16029572,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVulnerability of ALS mutant proteins to G4 oxidation. \u003c/strong\u003e(A) TDP-43, which is encoded by the \u003cem\u003eTARDBP\u003c/em\u003e gene, consists of two RRM with strong RNA-binding properties and a glycine-rich domain that regulates specific interactions. Ten mutations associated with patients with ALS, which were used in this study, are shown. The order/disorder propensity of TDP-43 was predicted using the Protein DisOrder prediction System (PrDOS) algorithm. (B) Purified TDP-43 wild-type and 10 mutant proteins used in this study. One microgram each of proteins was separated by 12.5% SDS-PAGE and detected by Coomassie brilliant blue staining. (C) Agarose gel mobility shift assay with \u003csub\u003eAx647\u003c/sub\u003ePSD-95-G4. Wild-type TDP-43 and the 10 mutant proteins were reacted with the probe in the presence or absence of competitor RNA (10% 8OG, 10-fold for probe). (D) Each overshifted band was quantified, and the mean and standard error (± SEM) obtained from three independent experiments were calculated and shown in the graph. \u003cem\u003eE\u003c/em\u003e, Graph showing the average binding with and without competitor RNA. The ±SEM from three independent experiments is displayed. Statistical significance was assessed using a two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"AIFig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7634186/v1/d6de6470c4f779766adaa86f.jpg"},{"id":102785526,"identity":"f955b34a-4b65-4af8-8330-ab699e47f365","added_by":"auto","created_at":"2026-02-16 16:07:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":50702220,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7634186/v1/c79b9b35-b16e-4c24-a5cb-45f3f883408e.pdf"},{"id":93744407,"identity":"c255eda7-d85a-440e-b5cb-26e0aa979890","added_by":"auto","created_at":"2025-10-17 06:11:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1898294,"visible":true,"origin":"","legend":"","description":"","filename":"AI8OGSUP2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7634186/v1/fcc72d47aa5e6561e5465b36.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Impact of G-quadruplex RNA oxidation on its conformational dynamics and interaction with ALS-associated TDP-43","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAmyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the selective loss of upper and lower motor neurons [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, the cause of the selective vulnerability of motor neurons remains unknown, which hinders the development of effective treatments. Approximately 50 ALS-associated and possibly related genes have been identified, most of which encode RNA-binding proteins (RBPs), such as TDP-43 (43-kDa TAR DNA-binding protein, encoded by \u003cem\u003eTARDBP\u003c/em\u003e) and fused in sarcoma (FUS) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These RBPs normally contribute to RNA metabolism and intra-axonal mRNA transport via RNA granules formed by liquid\u0026ndash;liquid phase separation (LLPS) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. mRNA recognition is highly selective, and guanine quadruplex (G4) is utilized as a common protein- binding signal [\u003cspan additionalcitationids=\"CR4 CR5 CR6\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. G4s are noncanonical DNA/RNA structures in which four guanines incorporate alkali metal ions by hydrogen bonding and fold over to stabilize them [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Therefore, the dysregulation of intra-axonal mRNA transport caused by abnormalities in G4-protein interaction can increase the risk of disease onset [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In support of this hypothesis, ALS-associated TDP-43 and FUS mutant proteins showed reduced binding compared with the wild-type in G4-binding assays [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In addition, considering cases where imperfections on the G4 side effect the interaction and cause abnormalities in mRNA transport is necessary.\u003c/p\u003e\u003cp\u003eAging is the primary risk factor for ALS, affecting sporadic and familial cases, and the number of patients continues to increase alongside increasing life expectancy [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The age dependence of ALS onset has some possible reasons, with increased oxidative stress being considered as a major risk factor. Oxidative stress arises because of an excess of reactive oxygen species (ROS), such as superoxide radicals, hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), and hydroxyl radicals; such ROS are generated as metabolic by products by biological systems [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Over the last 30 years, the causal relationship between ALS and oxidative stress has been investigated, with most studies targeting oxidative damage to DNA and proteins. However, no ALS-associated gene mutations that are involved in the repair of oxidized DNA and proteins have been reported, and most of the mutations occur in genes that encode for RBPs, axonal transport-related proteins, and redox-active proteins [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Furthermore, oxidative damage accumulates preferentially in the RNA pool over DNA and proteins [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Compared with DNA, which is double stranded, bound to histones, and compartmentalized in the nucleus, RNA has been reported to be 14\u0026ndash;25 times more susceptible to oxidation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In recent years, age-related RNA oxidation has received considerable attention, particularly in relation to neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and ALS [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Among nucleotides, guanine has the lowest redox potential, and it is easily oxidized. Therefore, G4-RNA composed of guanine nucleotides is highly sensitive to oxidative stress [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Substituting guanine with an oxidized form, 8-oxoguanine (8OG), leads to the rearrangement of hydrogen bond donors and acceptors on the Hoogsteen ends of the nucleobase, affecting the cation localization and exchange properties [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The accumulation of 8OG has been reported in aged neurons, ALS patients, and animal models [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Of the ALS-associated and possibly related genes identified, 19 code for RBPs and RNAs [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. All of these genes encode G4-binding proteins (G4BPs), G4-regulatory proteins, or abundant G4-forming sequences, raising concerns about a link between ALS and guanine oxidation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Although the destabilization of G4s by oxidative modifications has been primarily studied as a mechanism for the switch-on of oxidative stress\u0026ndash;responsive G4-containing promoters and mRNAs [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], whether the loss of conformation affects interactions with G4BPs has not been demonstrated.\u003c/p\u003e\u003cp\u003eRNA oxidation does not result from neuronal cell death, but an early event associated with pathogenic mechanisms [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Consequently, redefining this dysregulated process is essential. In this study, G4-RNAs containing 8OG substitutions were used, which mimic age-related oxidative damage, to analyze its comprehensive conformational changes and interactions with TDP-43, a G4BP with high binding specificity. This study, which focuses on the relationship between G4-RNA oxidation and ALS-associated G4BP, will provide novel insights into the molecular mechanism of age-related ALS onset.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eG4 conformation is affected by oxidative modifications\u003c/h2\u003e\u003cp\u003eFirst, immunocytochemistry was performed using the human neuroblastoma cell line GOTO to visualize the effect of oxidative stress on G4-RNA/DNAs in cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The cells were treated with hydrogen peroxide for 30 min [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Then, the medium was exchanged, and the cells were fixed 12 h later and probed with the anti-G4 antibody BG4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Fluorescence images showed that cells treated with hydrogen peroxide had reduced the G4 signals compared with untreated controls. However, this result does not necessarily lead to the conclusion that oxidative modification of G4 caused the conformational change. G4BPs bind to entire G4, making them difficult to detect adequately with antibodies that have conformational binding properties [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Fay et al. proposed the classification of G4BPs into those that bind and prevent the structure (Group 1; FUS; α-synuclein; zinc finger protein 106, Zfp106 etc.) and those that stabilize the structure (Group 2; TDP-43; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Both groups can exhibit protection from recognition by anti-G4 antibodies. In support of this hypothesis, the fluorescence images showed that the G4 and TDP-43 signals barely overlapped (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). In cells, the number of foci detected by anti-G4 antibodies is orders of magnitude lower than the number of G4-forming sequences predicted genome wide [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Hence, total RNAs from stressed cells were purified and probed using the G4-specific fluorophore, Thiazole orange biotin (TO1B) to quantitatively evaluate the effect of oxidative modifications on G4-RNA [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. As a result, the fluorescence intensity of TO1B decreased in a hydrogen peroxide concentration-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). This result is due to conformational alterations of G4-RNAs, excluding the effect of protein binding. Oxidative treatments in these cell-based assays may also affect transcription and RNA stability. Thus, precisely quantifying the effect of oxidative RNA modifications on the direct interaction of TDP-43 with G4s is necessary. Therefore, synthetic G4-RNAs derived from two types of mRNA 3\u0026prime;-UTR, namely, PSD-95 (postsynaptic density protein 95) and CaMKIIα (calcium/calmodulin-dependent protein kinase type II subunit alpha), were prepared [\u003cspan additionalcitationids=\"CR4 CR5 CR6\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B). Based on previous reports, these two mRNAs are transported to neurites in a G4-dependent manner and translated locally [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. These G4-RNAs were synthesized using phosphoramidites containing guanine mixed with 10%, 20%, or 40% 8OG to serve as oxidized model G4-RNAs (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The 8OG dose-dependent comprehensive modification of these synthetic model RNA mixtures was demonstrated by Western blotting using an anti-8OG antibody and electrophoresis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and D, Supplementary Fig. S2 and Fig. S3). Electrophoresis was performed under nondenaturing conditions, and staining with an intercalating fluorescent agent confirmed that the signal was remarkably reduced depending on the ratio of 8OG (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, and Supplementary Fig. S3). A slow mobility was observed for the most highly oxidized CaMKIIα-G4 (40% 8OG). The difference in staining levels may be due to a conformational change in G4, which inhibits the binding of the intercalating agent. 8OG destabilizes the structure by preventing the formation of Hoogsteen hydrogen bonds [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] while it has been reported to increase the stability [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eConformational alterations caused by guanine oxidation\u003c/h3\u003e\n\u003cp\u003eIn evaluating the conformational effects of model G4-RNAs with 8OG substitutions, Circular dichroism (CD) spectroscopy measurements were performed. RNA forms only parallel stranded G4s, and the CD spectrum shows a negative peak at around 240 nm and a positive peak at around 265 nm, which are a characteristic of the structure. However, the CD spectra of G4-RNAs derived from PSD-95 and CaMKIIα mRNAs showed different effects of the substitution with 8OG (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). CaMKIIα-G4 showed a decrease in positive and negative peaks in an 8OG dose-dependent manner. This result indicates that oxidative modifications of CaMKIIα-G4 structures often contribute to destabilization. In contrast, the spectrum of PSD-95-G4 was reduced at 40% 8OG, whereas both peaks were enhanced in RNA containing 10% and 20% 8OG, with the positive peak shifted slightly to the longer wavelength side (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The comparison of the CD spectra of PSD-95-G4 and RNAs with 8OG substitutions leads to two possibilities: (1) Substitution with 8OG stabilized the structure, or (2) the presence of a poly-A configuration led to an 8OG-adenine mismatch pair. Adenine frequently pairs with the Hoogsteen edge of 8OG, causing structural alteration [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The associated RNA regions that could be formed by the 8OG-A mismatch may enhance the spectrum close to the positive peak of G4. In investigating these possibilities, a fluorescent turn-on assay was performed using TO1B (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and D). The measurements showed that the fluorescence intensity of not only CaMKIIα-G4 but also PSD-95-G4 decreased in response to the rate of 8OG substitution. The decrease in TO1B signals in the 10% 8OG and 20% 8OG model RNA mixtures of PSD-95 indicated instability as a G4 structure, which might be due to an 8OG-A mismatch. The slight shift of the peak to longer wavelengths could be due to this effect [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eHence, in this study, detection was performed using naphthyridine\u0026ndash;azaquinolone (N-A), which binds to the G-A mismatch [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. N-A exhibits a UV absorption peak around 320 nm, which decreases upon binding to the G-A mismatch (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). The absorbance at 320 nm did not change for nonoxidized G4, but it decreased for all three types of oxidized G4s (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Under oxidative stress conditions, 8OG-A mismatch is observed in double-stranded DNA/RNA and codon\u0026ndash;anticodon interactions [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], which indicated that this phenomenon also occurs in G4-RNA. In addition, the decrease in the 320-nm peak caused by mismatch formation was more pronounced for the 10% substituted RNA than for the 20% and 40% substituted RNAs. The positive peak in the CD spectrum was also highest at 10% 8OG, indicating that a transient G4 conformation favors mismatch formation. Thus, mismatch formation does not depend on the substitution rate of 8OG but is further promoted by the stable proximity of adenine to 8OG through transient G4 formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). In addition, highly oxidized RNAs strongly inhibit the formation of the G4 structure, thereby reducing the accessibility of 8OG to A and suppressing mismatch formation. Furthermore, CaMKIIα-G4, which has a thermodynamically stable structure without an A-loop [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], is more sensitive to 8OG substitution, that is, changes in hydrogen bond positions, compared with PSD-95-G4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and D). These results imply that there are different types of G4 conformational changes upon guanine oxidation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG), and the combination of CD spectroscopy with other analytical techniques could avoid misinterpretations.\u003c/p\u003e\n\u003ch3\u003eG4 oxidation reduces TDP-43 binding caused by conformational alterations\u003c/h3\u003e\n\u003cp\u003eTo date, no analysis has been performed on the interaction between G4BP and G4 structures altered by 8OG substitution. Most G4BPs contain intrinsically disordered regions, making them difficult to purify and handle, and few proteins have been demonstrated to bind exclusively to G4s [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Previously, our group successfully purified full-length, tag-free TDP-43 and FUS, demonstrated their interactions with DNA/RNAs, and revealed differences in their binding specificity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. FUS binds to all three types of G4s (parallel, hybrid, and antiparallel) and a hairpin structure, whereas TDP-43 recognizes only parallel G4 and does not bind to a hybrid, antiparallel, and pUG fold [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] (Supplementary Fig. S4, Fig. S5, and Fig. S6). Therefore, TDP-43, which has high binding specificity, is suitable for measuring the interactions related to the G4 conformational alterations in this study.\u003c/p\u003e\u003cp\u003eThe extent to which the 8OG substitution affects the interaction through changes in the G4 structure was confirmed by a gel shift assay. The binding of TDP-43 to fluorescently labeled nonoxidized G4 was quantified by competition with unlabeled RNA containing 0, 10%, 20%, or 40% 8OG substitutions. These nonlabelled competitor RNAs were added at threefold and 10-fold the fluorescently labeled nonoxidized G4 probe level. The inhibitory effect of PSD-95-G4 decreased as the proportion of 8OG increased, and it was almost undetectable at 40% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and Supplementary Fig. S7). On the contrary, CaMKIIα-G4 was more sensitive to 8OG, and it showed no inhibitory effect even at 10% substitution (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and Supplementary Fig. S7), which was consistent with the results of the CD spectra and TO1B turn-on assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These results indicated that the oxidation of G4 induces conformational alterations that reduce the interaction with TDP-43. In addition, the effects of 8OG are not identical, but they vary greatly depending on the type of G4. PSD-95-G4 has a higher redundancy possibly because of the flexible A-loop, and it is more resistant to oxidation. By contrast, the dense and thermodynamically stable CaMKIIα-G4 is less redundant and is disruptively altered by the 8OG substitution. However, CaMKIIα-G4, which contains the most 8OG (40% 8OG), showed slight binding inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and Supplementary Fig. S7).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOur group has previously demonstrated that TDP-43 stabilizes the G4 structure as group 2 G4BP [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Therefore, in this study, CD spectroscopy was used to examine whether these conformational changes occurred upon the binding of oxidized PSD-95-G4s (10% and 20% 8OG) with TDP-43 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Upon binding to TDP-43, the structure of the nonoxidized G4 was barely altered [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], but the positive and negative peaks decreased for both oxidized RNAs. Therefore, the conformational alternation caused by the 8OG-A mismatch was suppressed by binding with TDP-43. Considering that TDP-43 cannot bind to RNA with a disrupted G4 structure, it should not repair the mismatch or rearrangement of the hydrogen bond. It likely binds to the transition intermediate G4 and prevents the formation of an 8OG-A mismatch (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e\n\u003ch3\u003eGuanine oxidation induces intermolecular G4 formation\u003c/h3\u003e\n\u003cp\u003eThe most highly oxidized CaMKIIα-G4 (40% 8OG) slightly but significantly inhibited protein binding. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In addition, this RNA was observed to exhibit retarded mobility upon electrophoresis under nondenaturing conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). It was hypothesized that molecules that cannot form intramolecular G4s might adopt intermolecular structures. In examining this hypothesis, asymmetric intermolecular G4 formation was attempted using the short G-rich RNA G\u003csub\u003e3\u003c/sub\u003eUG\u003csub\u003e3\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). This short RNA has only one loop-forming uracil, and symmetric intermolecular G4 structures are relatively unstable, and it also tends to form asymmetric intermolecular G4 structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. SYBR Green II staining showed an increase in CaMKIIα-G4 (40% 8OG) signal upon the addition of G\u003csub\u003e3\u003c/sub\u003eUG\u003csub\u003e3\u003c/sub\u003e, indicating the formation of asymmetric intermolecular G4 structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, C and D, and Supplementary Fig. S8). No synergistic effect was observed with the addition of G\u003csub\u003e3\u003c/sub\u003eUG\u003csub\u003e3\u003c/sub\u003e in PSD-95-G4 (40% 8OG). When lithium, which does not allow stable G4 formation, was used instead of potassium, no changes were observed upon the addition of G\u003csub\u003e3\u003c/sub\u003eUG\u003csub\u003e3\u003c/sub\u003e. In both RNAs with 20% 8OG substitutions, no increase in the signal caused by G\u003csub\u003e3\u003c/sub\u003eUG\u003csub\u003e3\u003c/sub\u003e could be detected (Supplementary Fig. S9 and Fig. S10). Therefore, the inhibition of protein binding by oxidized CaMKIIα-G4 (40% 8OG; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) was due to the promotion of intermolecular G4 formation. In this study, the formation of intermolecular G4s is likely detrimental to the regulation of mRNA transport. Consecutive guanine sequences are common in mRNAs, tRNAs, rRNAs, and ncRNAs. The abnormal intermolecular G4 formation with other G-rich RNAs may not only inhibit normal G4-mRNA transport but also lead to undesirable RNA metabolism and dysregulation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eG4 oxidation further increases the risk of ALS-associated TDP-43 mutations\u003c/h3\u003e\n\u003cp\u003eOur group previously investigated the effects of 10 ALS-associated amino acid substitution mutations in TDP-43 using surface plasmon resonance (SPR) analysis, demonstrating that all these mutations exhibit reduced interaction with G4-RNAs [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. TDP-43 interacts with G4s through three modules: two RNA recognition motifs (RRMs) with high affinity for RNA and a glycine-rich domain that modulates specific binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Most disease-associated mutations are located within the disordered glycine-rich region, indicating a link between binding specificity and disease onset [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The structural changes induced by G4 oxidation could pose a greater risk to the TDP-43 variants associated with ALS. To explore this finding, the 10 ALS-associated mutant proteins were used to analyze the effect of G4 oxidation on their interactions, using gel shift assays with a PSD-95-G4 probe and oxidized RNA competitor (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). The gel shift assays confirmed that all mutant proteins exhibited reduced binding to G4, which is consistent with previous SPR results [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, differences emerged in their inhibitory effects when competed with nonfluorescently labeled oxidized G4 (10%) compared with the wild-type (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and D, and Supplementary Fig. S11). Notably, all 10 mutants were less affected by unlabeled oxidized RNA than the wild-type (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE), indicating a weaker affinity for oxidized RNA. Among these, the G287S and P363A mutations, which showed the most significant difference from the wild-type, are located at the center of two peaks in the confidence values within the disordered regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). This phenomenon likely results from the combined effects of amino acid substitutions and conformational alterations of G4 within regions normally stabilized by G4 interactions. Overall, these findings indicate that the decreased interaction between ALS-associated TDP-43 mutants and G4-RNA is further exacerbated by guanine oxidation. However, at present, data regarding the earlier onset or shorter disease duration in patients with G287S and P363A mutations are limited. As more genotypic and phenotypic data are collected from patients, the causal relationship between \u003cem\u003eTARDBP\u003c/em\u003e mutations and age-related G4 oxidation will become more evident.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn recent years, oxidative RNA damage has gradually come to the forefront of attention in the areas of cancer, metabolic diseases, cardiovascular diseases, and neurodegenerative diseases [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. A variety of evidence, including studies of human and animal models, indicates that RNA oxidation is a hallmark of neurons in the aging brain and is more pronounced in neurons at the early stages of age-related neurodegenerative diseases [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Guanine has the lowest redox potential among nucleobases, and it is readily oxidized, leading to the formation of oxidized RNAs either through the direct oxidation of polynucleotide bases or the incorporation of oxidized bases during normal RNA synthesis by RNA polymerases [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Thus, G4s formed from four consecutive G regions were susceptible to oxidation, and the resulting conformational abnormality manifested as a reduced signal in cultured cells stained with an anti-G4 antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, although the conformational defects of G4-RNA caused by oxidation within cells could be quantified using extracted and purified RNA, detecting the interaction with binding proteins in cells was difficult. In enabling precise comparison and quantification, RNA mixtures were used in which guanines were randomly substituted with 8OG to mimic oxidation in cells, and the conformational changes of G4s caused by oxidative modifications, as well as their interactions with TDP-43, were analyzed. The effect of G4 oxidation varies depending on the sequence and loop configuration but comprehensively leads to a shift toward a less stable state, as quantitatively demonstrated by CD spectroscopy, and TO1B turn-on assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). One of the unexpected effects of oxidative modification on the G4 structure was the formation of an 8OG-A mismatch within the A-loop of PSD-95-G4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The mismatch formation was most efficient in RNA with 10% 8OG substitution rather than 20% or 40%, indicating that the proximity caused by G4 assembly is important to the formation. The decrease in the absorbance of mismatch-bound N-A and the inhibition of mismatch formation in the presence of TDP-43 also supported these predictions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Another unexpected effect was that the oxidation of guanine resulted in the formation of an intermolecular G4. Considering that misalignment of hydrogen bonds is fatal to the formation of intramolecular G4s, the remaining nonoxidized consecutive guanines form the intermolecular conformation. These guanines may even form abnormal G4s not only with various RNAs containing consecutive guanine sequences but also with DNA. Finally, the structural abnormalities caused by G4 oxidation led to reduced binding to TDP-43, and all 10 ALS-associated TDP-43 mutant proteins showed a reduced ability to bind to oxidized G4s compared with the wild-type (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These results imply that even low levels of oxidative damage to G4s can lead to dysregulation through synergistic effects with protein mutations.\u003c/p\u003e\u003cp\u003eRecently, a growing number of studies have reported that G4-RNAs are enriched within transient biomolecular condensates called stress granules, which are formed by LLPS as a cellular response to heat stress, endoplasmic reticulum stress, and oxidative stress, etc. [\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The enrichment of G4-RNAs and RBPs in stress granules is consistent with the aims of this study and is considered as an ideal molecular mechanism for protecting RNAs. Under short-term oxidative stress, RNA oxidation does not occur immediately and appears to be protected [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Furthermore, G3BP1 (Ras GTPase-activating protein binding protein 1) contributes to stress granule assembly, and its knockdown reduces stress granule size and impairs the preservation of mRNAs [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The dysfunction of G3BP1 could be involved in the onset of neurodegenerative diseases, including ALS, and G3BP1 is also a G4BP [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Thus, a system that preferentially enriches G4-mRNAs should be particularly neuroprotective, and dysregulation could pose risks to motor neurons, where the axonal transport of G4-mRNA is essential.\u003c/p\u003e\u003cp\u003eTo date, most studies on cellular damage caused by RNA oxidation have focused on the translation machinery. Oxidized RNAs cause miscoding and stalled ribosomes, leading to the production of abortive or defective peptides/proteins [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. 8OG-containing mRNA has been shown to reduce protein production and fidelity in cell-free translation systems and cultured cells [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Nonetheless, the findings indicate that the selective vulnerability of motor neurons to RNA oxidation in ALS should not be considered solely in terms of its effects on the translation machinery. Most of the genes causative for ALS encode RBPs, such as TDP-43, FUS, Senataxin (SETX), Zfp106, Angiogenin (ANG), Ataxin-2 (ATXN2), heterogeneous nuclear ribonucleoprotein RNA A1 (hnRNPA1), heterogeneous nuclear ribonucleoprotein A3 (hnRNPA3), hnRNPA2/B1 (heterogeneous nuclear ribonucleoprotein A2/B1), ewing sarcoma RNA-binding protein (EWSR1/EWS), T cell intracellular antigen-1 TIA1, RNA-binding motif protein 14 (RBM14), TATA-binding protein associated factor 15 (TAF15), and splicing factor proline and glutamine rich (SFPQ), all of which either directly bind to G4 or regulate G4-forming sequences [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In addition, the intronic hexanucleotide repeat expansion (GGGGCC)n in \u003cem\u003eC9ORF72\u003c/em\u003e (chromosome 9 open reading frame 72) that forms numerous G4 structures is the most common genetic cause of ALS [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. The effects of G4 oxidation confirmed in this study likely extend to all these factors, which could increase the risk of developing ALS. G4-dependent long-distance mRNA transport is more important in motor neurons than in other cells [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Therefore, motor neurons suffer most from the dysregulation of G4-dependent long-distance mRNA transport. Collectively, the vulnerability of G4s to oxidation leads to diverse conformational alternations and reduced binding to proteins involved in axonal mRNA transport. This effect may be most pronounced in neurons, indicating that G4-RNA oxidation is a molecular hallmark of aging that contributes to increased ALS risk.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eCell culture\u003c/h2\u003e\u003cp\u003eThe GOTO human neuroblastoma cell line (Japanese Collection of Research Bioresources, JCRB0612) was cultured in RPMI 1640 medium (Thermo Fisher Scientific Inc. Waltham, Massachusetts, USA) supplemented with 10% fetal calf serum and 1% penicillin\u0026ndash;streptomycin (Thermo Fisher Scientific Inc. Waltham, Massachusetts, USA) at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e\u003cp\u003eImmunofluorescence staining of cultured cells was performed as previously described\u003csup\u003e4\u003c/sup\u003e, using a rabbit polyclonal anti-TDP-43 antibody (Cell Signaling Technology Inc., #3449 Danvers, Massachusetts, USA) and the mouse monoclonal anti-G-quadruplex antibody BG4 (Absolute Antibody Ltd., Ab00174-1.1 Redcar, UK).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eThiazole orange biotin (TO1B) turn-on assay\u003c/h2\u003e\u003cp\u003eTotal cellular RNAs were extracted using the TRIzol reagent (Thermo Fisher Scientific Inc. Waltham, Massachusetts, USA) in accordance with the manufacturer's instructions. Cellular RNA (20 \u0026micro;g) or synthetic RNA (20 pmol) was dissolved in 180 \u0026micro;L of buffer (20 mM PIPES-KOH, pH 6.8, 0.1 mM MgCl\u003csub\u003e2\u003c/sub\u003e, and 150 mM KCl), then mixed with 20 \u0026micro;L of 1 \u0026micro;M thiazole orange-3PEG biotin (TO1B; Applied Biological Materials Inc. Richmond, British Columbia, Canada), dissolved in the same buffer, and incubated at 25\u0026deg;C for 5 min. Afterward, ligand-enhanced fluorescence spectra were measured using a fluorescence spectrometer (Shimadzu RF-5300PC, Shimadzu Corporation) with an excitation wavelength of 505 nm and emission spectra ranging from 520 to 600 nm [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eRNA/DNA\u003c/h2\u003e\u003cp\u003eThe synthesized oligonucleotides, which were used in this study, were obtained from GeneDesign, Inc., and FASMAC Co., Ltd. (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eRNA staining\u003c/h2\u003e\u003cp\u003eIn staining dot-blotted RNAs on the membrane, the membrane was immersed in a 0.05% toluidine blue solution (Fujifilm Co., Ltd.) for 20 s and then washed with water. RNA separated by electrophoresis on an agarose gel under native conditions was stained with SYBR Green II (Takara Bio Inc.) in accordance with the manufacturer's instructions and visualized using the Typhoon 9410 imaging system (GE Healthcare).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eImmunoblotting\u003c/h2\u003e\u003cp\u003eRNAs (2.5 pmol) were blotted onto a nylon membrane (Roche Co., Ltd. Basel, Switzerland) and cross-linked using an Ultraviolet (UV) cross-linker (Funakoshi Co., Ltd. Tokyo, Japan). Immunoblotting was performed using an anti-8-oxo-guanine (8-oxoG) mouse monoclonal antibody (Merck Biopharma Co., Ltd.; SAB5200010 Tokyo, Japan) and an anti-mouse IgG-HRP antibody (Jackson Immuno Research Laboratories, Inc.; 715-035-151 West Grove, Pennsylvania, USA) to confirm guanine oxidation. Furthermore, signals were visualized and quantified using the Typhoon 9410 imaging system.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eCircular dichroism (CD) spectral analysis\u003c/h2\u003e\u003cp\u003eThe estimated G4 structures of the synthesized RNAs were confirmed by CD spectral analysis using a 2 \u0026micro;M oligonucleotide in a buffer containing 50 mM PIPES (sodium 2-[4-(2-sulfoethyl)piperazin-1-yl] ethanesulfonate)\u0026ndash;KOH (pH 6.8), 150 mM KCl, and 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e at 25\u0026deg;C, as previously described [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], with a spectropolarimeter (JASCO J-820). Before measurement, each RNA sample was heated at 98\u0026deg;C for 5 min and then allowed to cool to room temperature. The scans were repeated five times, and Jasco Spectro Manager software was used to compile the average spectra. The x-axis represents the wavelength, while the y-axis indicates the molar CD (Δε). For binding experiments with the TDP-43 protein, an additional final concentration of 1.25% glycerol, prepared from the protein stock solution, was included.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eUV absorption measurement of naphthyridine\u0026ndash;azaquinolone (N-A) hybrids\u003c/h2\u003e\u003cp\u003eBefore use, each oxidized RNA sample (6 \u0026micro;M) prepared in a buffer containing 20 mM PIPES-KOH (pH 6.8), 150 mM KCl, and 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e (40 \u0026micro;L) was heated at 98\u0026deg;C for 3 min and then cooled to room temperature. Subsequently, 10 \u0026micro;L of the 100-\u0026micro;M N-A solution (Synthonix Inc.; AC80704) prepared in buffer containing 0.1% (w/v) dimethyl sulfoxide was added to the sample. After incubation for 15 min at room temperature, absorption was measured using a spectrophotometer (Hitachi High-Tech Co., Ltd.; U-2800).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eTDP-43\u003c/h2\u003e\u003cp\u003ePurified, no-tag, full-length dimeric forms of recombinant human TDP-43 (wild-type and mutant proteins) derived from \u003cem\u003eEscherichia coli\u003c/em\u003e were prepared as previously described [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The protein storage buffer contained 10% glycerol, 20 mM PIPES-KOH (pH 6.8), and 150 mM KCl. The wild-type protein was confirmed to exhibit the same binding activity and specificity for G4 structures as the protein was immunopurified from human cultured cells [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eGel shift assay\u003c/h2\u003e\u003cp\u003eThe gel shift assay was performed as previously described [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Fluorescently labeled oligonucleotides at the 5\u0026prime;-end, including \u003csub\u003eAx647\u003c/sub\u003ePSD-95-G4 and \u003csub\u003eAx647\u003c/sub\u003eCaMKIIα-G4 (0.5 pmol each), were incubated with TDP-43 (2 pmol) in a 10-\u0026micro;L reaction buffer (containing 10% glycerol, 20 mM PIPES\u0026ndash;NaOH, pH 6.8, 1 mM MgCl₂, and 150 mM KCl) in the absence or presence of competitor RNAs. The buffer also contained 0.1 mg/mL bovine serum albumin and 0.05 mg/mL poly-dI-dC (Thermo Fisher Scientific Inc.). Incubation was performed at 25\u0026deg;C for 30 min. Then, the samples were loaded onto a 0.5\u0026times; TBE (Tris-Borate-EDTA; pH 6.8) buffer containing 1% agarose gel and electrophoresed at 50 V for 60 min at 25\u0026deg;C. The gels were visualized and quantified using the Typhoon 9410 imaging system, as previously described [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eIntermolecular G4 formation\u003c/h2\u003e\u003cp\u003eThe oxidized model G4-RNAs (20 pmol) were mixed with the absence or presence of G\u003csub\u003e3\u003c/sub\u003eUG\u003csub\u003e3\u003c/sub\u003e (20 pmol) in a 10-\u0026micro;L buffer containing 20% glycerol, 20 mM PIPES\u0026ndash;NaOH (pH 6.8), 1 mM MgCl₂, and 150 mM KCl or 150 mM LiCl. The samples were heated at 98\u0026deg;C for 5 min and then cooled to room temperature. Each sample was loaded onto a 1% agarose gel containing 0.5 \u0026times; TBE and electrophoresed at 50 V for 60 min at 25\u0026deg;C. The gels were stained with SYBR Green II and analyzed using Molecular Imager FX (Bio-Rad Laboratories, Inc. Hercules, California, USA).\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor contributions statement\u003c/h2\u003e\n\u003cp\u003eConceptualization, investigation, data curation, visualization and writing: AI\u003c/p\u003e\n\u003ch2\u003eCompeting interest\u003c/h2\u003e\n\u003cp\u003eThe author declares no competing interests.\u003c/p\u003e\n\u003ch2\u003eSupporting information\u003c/h2\u003e\n\u003cp\u003eThis article contains supporting information.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research (22K07032, 23H04265 and 25K10307) and SERIKA FUND to AI.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eConceptualization, investigation, data curation, visualization and writing: AI\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eI thank Dr. M. Ishiguro for support and helpful advice.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe data sets generated during and/or analyzed during the current study are available from the corresponding authors on reasonable request. ([email protected]).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBoill\u0026eacute;e, S., Velde, V., Cleveland, D. W. \u0026amp; C. \u0026amp; ALS: a disease of motor neurons and their nonneuronal neighbors. \u003cem\u003eNeuron\u003c/em\u003e \u003cb\u003e52\u003c/b\u003e, 39\u0026ndash;59 (2006).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTaylor, J. P., Brown, R. H. Jr \u0026amp; Cleveland, D. W. Decoding ALS: from genes to mechanism. \u003cem\u003eNature\u003c/em\u003e \u003cb\u003e539\u003c/b\u003e, 197\u0026ndash;206 (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIshiguro, A. \u0026amp; Ishihama, A. 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PrDOS: prediction of disordered protein regions from amino acid sequence. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/nar/gkm363\u003c/span\u003e\u003cspan address=\"10.1093/nar/gkm363\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2007).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"RNA, 8-Oxoguanine (8‐oxoG), TAR DNA‐binding protein 43 (TDP‐43) (TARDBP), amyotrophic lateral sclerosis (ALS) (Lou Gehrig disease), G‐quadruplex, aging","lastPublishedDoi":"10.21203/rs.3.rs-7634186/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7634186/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAmyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the selective degeneration of motor neurons. The primary cause of ALS, whether sporadic or familial, is aging, and recent studies have shown that age-related RNA oxidation plays a role in the early stages of disease onset. This study focused on the vulnerability of G-quadruplex (G4) structures to oxidation and aimed to elucidate the molecular mechanism underlying the conformational changes and their interactions with the binding protein TDP-43. Guanine within G4 structures has a low redox potential, and its substitution with 8-oxoguanine (8OG) can induce structural instability and impair its function as a protein binding signal. In addition, synthetic G4-RNAs modified by oxidation were examined, and results showed that conformational changes are due to different hydrogen bond arrangements, 8OG-A mismatches, and intermolecular G4 formation. The interaction between G4 and TDP-43 decreased in proportion to the substitution rate of 8OG. Furthermore, ALS-associated mutant proteins exhibited reduced binding affinity for oxidized G4s compared with the wild-type. Considering that intra-axonal mRNA transport mediated by G4-binding proteins is essential for the survival and activity of motor neurons, this study will provide important insights into the molecular mechanisms underlying the onset of ALS with aging.\u003c/p\u003e","manuscriptTitle":"Impact of G-quadruplex RNA oxidation on its conformational dynamics and interaction with ALS-associated TDP-43","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-17 06:11:04","doi":"10.21203/rs.3.rs-7634186/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-29T19:23:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-23T09:58:52+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-20T02:21:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-14T11:36:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"126831921323142113923143709576332542310","date":"2025-10-08T12:39:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"194261294167787336593530474489362151886","date":"2025-10-07T01:01:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"220829171048034714967899376490308596493","date":"2025-10-06T23:57:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-06T23:40:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-06T11:24:41+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-23T09:25:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-19T00:55:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-09-19T00:48:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a93e59e9-b04b-47ed-a623-60fd891bf50b","owner":[],"postedDate":"October 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":56273024,"name":"Biological sciences/Biochemistry"},{"id":56273025,"name":"Biological sciences/Biophysics"},{"id":56273026,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2026-02-16T16:04:56+00:00","versionOfRecord":{"articleIdentity":"rs-7634186","link":"https://doi.org/10.1038/s41598-026-39767-y","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-02-13 15:57:33","publishedOnDateReadable":"February 13th, 2026"},"versionCreatedAt":"2025-10-17 06:11:04","video":"","vorDoi":"10.1038/s41598-026-39767-y","vorDoiUrl":"https://doi.org/10.1038/s41598-026-39767-y","workflowStages":[]},"version":"v1","identity":"rs-7634186","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7634186","identity":"rs-7634186","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}