The bacterial pore-forming toxin α-hemolysin has dimensions appropriate for capture and translocation of DNA strands in a single-molecule electrophoresis experiment. We used the nanopore’s properties to study G-quadruplex unfolding in the human telomere repeat sequence with and without the presence of DNA lesions introduced into either the GGG tracks of a potential G-quadruplex (via oxidation) or the TTA loops (via photodimerization). Different topological folds of the G-quadruplex could be distinguished by either their current-time signatures or by their unfolding rates when a dA25 tail was added. Another more compact four-stranded structure, the i-motif, was also studied and found to be exceptionally well folded and stable inside the nanopore cavity. Comparisons are made between early studies with a home-built nanopore device vs. the currently available instrument from Oxford Nanopore Technologies (ONT), showing that we approach, but do not yet achieve, single-molecule sequencing of DNA damage sites in human telomere repeats. These studies aid in our understanding of the structure and dynamics of non-canonically folded DNA, its behaviour in crowded environments that mimic intracellular conditions, and the ability to use nanopore sequencing to identify DNA damage sites in this oxidation-prone segment of the genome.

The iron-Fenton reaction in biology is influenced by diverse cellular molecules that coordinate with redox-active ferrous ion. Contemporary research proposed that hydroxyl radical or a ferryl species (HO•/Fe=O2+) are the primary oxidants; however, this was challenged by the observation that physiological bicarbonate redirects the reaction to form carbonate radical anion (CO3•-). Questions remained regarding the roles of O2 concentration, ascorbate, and iron speciation in CO3•- formation. Accordingly, in cellulo studies were conducted under physiological O2 (∼25 μM) with ascorbate replenishment to monitor bicarbonate-dependent telomeric DNA damage. Under these conditions,. physiological bicarbonate (25 mM) yielded 2'-deoxyguanosine-specific oxidation consistent with CO3•- formation at a ratio exceeding 80:1 relative to HO•/Fe=O2+. In parallel in vitro experiments using a plasmid nicking assay, the cellular low molecular weight (LMW) ultrafiltrate was used as the source of iron and its endogenous coordination partners; under physiological O2, bicarbonate, and 500 nM H2O2 (the concentration required to produce detectable signal), the DNA damage profile was consistent with exclusive CO3•- formation, mirroring the cell culture result. A panel of iron complexes approximating the intracellular labile iron pool (5 μM) was examined: hexaaquo-ferrous ion, ferrous citrate, ferrous α-ketoglutarate, ferrous pyrophosphate, ferrous glutathione, and hemin. Of these, only hemin reproduced the bicarbonate-dependent CO3•- damage profile observed in cells with 100 nM H2O2 and 25 mM bicarbonate present. This finding was corroborated using a defined biomimetic metabolome in which hemin, ferrous ion, or their combination was tested; hemin consistently supported CO3•- as the dominant oxidant. Roles for the Udenfriend reaction (Fe(II), O2, and reductant) and superoxide dismutase were also studied. Collectively, these results identify heme iron as a likely candidate to drive CO3•- formation via the bicarbonate iron-Fenton reaction to damage dG in DNA during endogenous oxidative stress.

Chemical damage to ribosomal RNA (rRNA) during oxidative or inflammatory stress can impact protein synthesis. Human cells were exposed to an H2O2 titration to induce oxidative stress, or tumor necrosis factor-α (TNF-α) over a time course to induce inflammation, followed by direct nanopore RNA sequencing of cytosolic and mitochondrial rRNAs to reveal damage sites. Guanosine (G) oxidation and deamination of adenosine to inosine (A-to-I) and cytidine to uridine (C-to-U) were identified by changes in the base-called data. Both stressors induced G oxidation in cytosolic rRNA, whereas mitochondrial rRNA was less oxidatively modified. Nitrosative stress during inflammation caused deamination lesions in rRNAs in both compartments. Inspection of highly modified sites found that GC-rich tentacles in the 28S rRNA expansion sequences were hotspots for G oxidation and C deamination in the cytosolic ribosome. Outside of tentacles, lesions generally occurred on surface nucleotides. The minimalist mitochondrial ribosome structure, compared to the cytosolic ribosome, alters reaction patterns such that nucleotides on the surface or in functionally relevant regions are damaged. These patterns suggest that tentacles on cytosolic rRNA direct reactive oxygen and nitrogen species away from the catalytic core to maintain activity during stress, while the mitochondrial ribosome is damaged in regions that can deactivate protein synthesis. These results provide molecular insight into metabolic dysfunction and suggest a new function for the GC-rich tentacles evolved in mammalian cells.

Ribosomal RNA (rRNA) modifications are tuned to regulate protein synthesis; however, their temporal dynamics during oxidative or inflammatory stress remain poorly understood. Nanopore direct RNA sequencing using Dorado v5.2.0 modification-aware models for the data analysis was employed to map human rRNA epitranscriptomic marks in a cell line undergoing oxidative stress, inflammatory stress, or ferroptosis. Oxidative stress triggered a global trend of decreased modification occupancy in which six modifications shifted significantly over 48 h, particularly, 18S Ψ573 and 18S m6A1832. Conversely, inflammatory stress induced a complex response involving an acute pulse of hypermodifications at 28S Um1773 and 28S Ψ1779, for example, and chronic hypomodification at specific target sites (e.g., 18S Gm1328 and 28S Gm4228). In this work, the pseudouridine modifications 28S Ψ4296 and 28S Ψ4353 were identified as “universal stress markers” that decreased under all stressors studied, including ferroptosis. Mapping these changes onto the ribosome structure revealed that they reside in functional regions such as the decoding center and A-site finger, supporting a role in functional ribosome reprogramming during stress. Analysis of mitochondrial rRNA (mt-rRNA) revealed modification shifts within the peptidyl transferase center, suggesting a mechanism to attenuate mitochondrial translation during chronic stress. This work demonstrates that oxidative and inflammatory stress drive distinct, time-resolved remodeling of the human rRNA epitranscriptome and provides a framework for using rRNA modifications as biomarkers of cellular health during oxidative or inflammatory stress exposure.

Nanopore direct RNA sequencing (DRS) is revolutionizing our ability to analyze the epitranscriptome to evaluate nucleoside modifications in both cellular and synthetic RNA. The process involves minimal handling of fragile RNA strands, one round of reverse transcription to provide a DNA:RNA duplex, and library preparation to directly read nucleotides with their modifications as they pass through a protein nanopore embedded in a membrane. Simultaneous sequencing of hundreds of strands on a chip provides unprecedented access to whole transcriptome information. A key advantage is the long-read length that permits, for example, operon-specific epitranscriptomics of ribosomal RNA modifications as a function of cellular stress. By analyzing the entire transcriptome, the interplay of different modifications on the same RNA, or the correlation of changes in different RNAs in the same cell type, can be monitored. This review presents several recent examples of the types of experiments that are suitable for nanopore DRS as well as some of the current challenges and future expectations.

Apurinic/apyrimidinic endonuclease-1 (APE1) is a repair enzyme that efficiently cleaves abasic (AP) site damage in duplex DNA. Reports of in vitro activity assays between APE1 and single-stranded G-quadruplex (ssG4) reveal significant decreases in the endonuclease activity. Here, we identify that the low yields observed represent cleavage of the noncanonical folds that did not adopt a complete G4 fold. This conclusion is supported through circular dichroism analysis and activity assays analyzing the cleavage rate, folding impact on cleavage, and product inhibition. Studies were performed on AP-containing ssG4 and duplex-embedded G4 scaffolds. The CD spectra of a non-G4 containing potential quadruplex sequence reveal a noncanonical structure. APE1 can cleave an AP in these non-G4 conformation(s) in high yields comparable to the preferred duplex substrate. There is direct evidence of decreasing APE1 activity with increasing G4 folding in ssG4 and duplex-G-quadruplex-duplex (DGD) systems. Also observed is a positional dependency on yield in the non-G4 DGD scaffolds, but not in the non-G4 ssG4 scaffolds. In conclusion, our studies provide evidence that APE1 efficiently cleaves noncanonical conformations in G4-like structures, highlighting the control of secondary structure on APE1 endonuclease activity.

Telomere dysfunction and attrition are observed in cells subjected to oxidative or inflammatory stress, largely due to DNA damage. In this study, we applied a DNA glycosylase-assisted quantitative PCR (qPCR) assay to assess telomeric DNA damage in human cells exposed to defined sources of oxidative or inflammatory stress. As a consequence of the endogenous chemistry of these stressors being different, we were able to identify distinct types of telomere DNA damage that differentiate oxidative from inflammatory stress. By selecting lesion-specific DNA glycosylases before qPCR analysis, we determined that reactive oxygen species generated under physiological bicarbonate buffering during both oxidative and inflammatory stress primarily damaged 2'-deoxyguanosine (dG) residues. In contrast, inflammatory stress increased dG oxidation sites and nitrosative DNA damage, evidenced by deamination of 2'-deoxycytosine (dC) to 2'-deoxyuridine (dU) and 2'-deoxyadenosine (dA) to 2'-deoxyinosine (dI; hypoxanthine). During inflammation, telomeric DNA contained 1.5-fold more dU than dI. These nitrosative lesions require different DNA repair enzymes compared with dG oxidation products, and they may impact telomere structure differently. Our findings suggest that beyond dG oxidation, nitrosative DNA damage can be a major source of lesions in telomeres. The profile of telomere DNA lesions can serve as a biomarker to distinguish between oxidative and inflammatory stress.

Oxidative damage to RNA is associated with neurodegeneration, cardiovascular diseases, and cancer development. Studies that monitor RNA damage by H2O2 often omit the physiological buffer bicarbonate in the reaction, which fails to account for the influence of the buffer on the iron-Fenton reaction. Herein, we monitored two in vitro systems to understand how bicarbonate redirects the iron-Fenton reaction from a hydroxyl radical (HO•) generator in the absence of bicarbonate to one that predominantly yields carbonate radical anion (CO3•-) in the presence of this buffer. Using the HO•-selective fluorophore terephthalic acid, we found that the Fe(II)-ligand identity impacted the bicarbonate concentration required to transition the Fenton reaction to predominantly yield CO3•-. These findings were then corroborated by following the oxidation of guanosine (rG), which reports on oxidation by both radicals, and uridine (rU) oxidation, which responds to only HO• as the oxidizing species. The studies found that as the Fe(II)-ligand complex stability increased, the bicarbonate concentration inflection point to favor CO3•- production and rG oxidation also increased. Regardless of the ligand strength, the crossover values obtained were below physiologically relevant bicarbonate concentrations (<20 mM). Next, Escherichia coli or HEK293T cells were pre-equilibrated with bicarbonate from 0 to 20 mM before a bolus addition of H2O2. The bicarbonate-dependent inflection points for favoring CO3•- over HO• (or ferryl) for E. coli (7.3 mM) and HEK293T (11.3 mM) cells differed, but were below physiologically relevant concentrations, supporting the hypothesis that the cellular iron-Fenton reaction normally yields CO3•-. The redox-cycling compound menadione was used for continuous in-cell generation of H2O2 to find bicarbonate dependencies in oxidation reactions of RNA. The studies herein point toward the redirection of the iron-Fenton reaction in cells to predominantly yield CO3•- that selectively damages rG sites in the transcriptome.

Antimicrobial resistance (AMR) threatens global health; however, the molecular adaptations underlying resistance to emerging antibiotic classes remain poorly defined. Here, we applied long-read DNA and direct RNA nanopore sequencing and developed methods to deconvolute operon-specific epitranscriptomic changes. Together, this platform uncovered a previously unrecognized, operon-specific pathway of resistance in Staphylococcus aureus to the naphthyridone antibiotic A-692345. Genomic nanopore sequencing identified a single 23S rRNA mutation (T1732C) confined to one of the six rRNA operons (operon 2), which uniquely contains nine tRNA genes. RNA direct nanopore sequencing generated a comprehensive and updated rRNA modification map for S. aureus and revealed extensive remodeling of rRNA modifications in the resistant strain. Differentially incorporated modifications included pseudouridine, dihydrouridine, and 5-hydroxycytidine at functionally relevant positions within the ribosome. Upon mapping these epitranscriptomic changes, we noted they were operon specific. This likely gives rise to ribosome heterogeneity with potential for selective translation of stress-response genes that favor resistance. Collectively, these findings establish nanopore sequencing as a powerful platform for resolving coupled genomic and epitranscriptomic adaptations, providing molecular insight into how bacteria evolve resistance to antibiotics.

In the last decade, several novel functions of the mammalian Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APE1) have been discovered, going far beyond its canonical function as DNA repair enzyme and unveiling its potential roles in cancer development. Indeed, it was shown to be involved in DNA G-quadruplex biology and RNA metabolism, most importantly in the miRNA maturation pathway and the decay of oxidized or abasic miRNAs during oxidative stress conditions. In recent years, several noncanonical pathways of miRNA biogenesis have emerged, with a specific focus on guanosine-rich precursors that can form RNA G-quadruplex (rG4) structures. Here, we show that several miRNA precursors, dysregulated upon APE1 depletion, contain an rG4 motif and that their corresponding target genes are up-regulated after APE1 depletion. We also demonstrate, both by in vitro assays and by using different cancer cell lines, that APE1 can modulate the folding of an rG4 structure contained in pre-miR-92b, with a mechanism strictly dependent on lysine residues present in its N-terminal disordered region. Furthermore, APE1 cellular depletion alters the maturation process of miR-92b, mainly affecting the shuttling between the nucleus and cytosol. Bioinformatic analysis of APE1-regulated rG4-containing miRNAs supports the relevance of our findings in cancer biology. Specifically, these miRNAs exhibit high prognostic significance in lung, cervical, and liver tumors, as suggested by their involvement in several cancer-related pathways.

Hydrogen peroxide is a precursor to reactive oxygen species (ROS) in cells because of its high reactivity with iron(II) carbonate complexes formed in the labile iron pool due to a high concentration of intracellular bicarbonate (25-100 mM). This chemistry leads to the formation of carbonate radical anion rather than hydroxyl radical, and unlike the latter ROS, CO3•- is a milder one-electron oxidant with high specificity for guanine oxidation in DNA and RNA. In addition to metabolism, another major source of DNA oxidation is inflammation which generates peroxynitrite, another precursor to CO3•- via reaction with dissolved CO2. The identity of the ROS is important because not all radicals react with DNA in the same way. Whereas hydroxyl radical forms adducts at all four bases and reacts with multiple positions on ribose leading to base loss and strand breaks, carbonate radical anion is focused on guanosine oxidation to yield 8-oxo-7,8-dihydroguanosine in nucleic acids and the nucleotide pool, a modification that can function epigenetically in the context of a G-quadruplex. DNA sequences of multiple adjacent guanines, as found in G-quadruplex-forming sequences of gene promoters, are particularly susceptible to oxidative damage, and the focusing of CO3•- chemistry on these sites can lead to a transcriptional response during base excision repair. In this pathway, AP-endonuclease 1 plays a key role in accelerating G-quadruplex folding as well as recruiting activating transcription factors to impact gene expression.

While hydroxyl radical is commonly named as the Fenton product responsible for DNA and RNA damage in cells, here we demonstrate that the cellular reaction generates carbonate radical anion due to physiological bicarbonate levels. In human and Escherichia coli models, their transcriptomes were analyzed by RNA direct nanopore sequencing of ribosomal RNA and chromatography coupled to electrochemical detection to quantify oxidation products in order to follow the bicarbonate dependency in H 2 O 2 -induced oxidation. These transcriptomic studies identified physiologically relevant levels of bicarbonate focused oxidation on the guanine base favorably yielding 8-oxo-7,8-dihydroguanine (OG). In human cells, the bicarbonate-dependent oxidation was further analyzed in the metabolome by mass spectrometry, and a glycosylase-dependent qPCR assay to quantify oxidation sites in telomeres. These analyses further identify guanine as the site of oxidation when bicarbonate is present upon H 2 O 2 exposure. Labile iron as the catalyst for forming carbonate radical anion was demonstrated by repeating the bicarbonate-dependent oxidations in cells experiencing ferroptosis, which had a >fivefold increase in redox-active iron, to find enhanced overall guanine-specific oxidation when bicarbonate was present. The complete profiling of nucleic acid oxidation in the genome, transcriptome, and metabolome supports the conclusion that a cellular Fe(II)-carbonate complex redirects the Fenton reaction to yield carbonate radical anion. Focusing H 2 O 2 -induced oxidative modification on one pathway is consistent with the highly evolved base excision repair suite of enzymes to locate G-oxidation sites for repair and gene regulation in response to oxidative stress.

In the last decade, several novel functions of the mammalian Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APE1) have been discovered, going far beyond its canonical function as a DNA repair enzyme, unveiling its potential roles in cancer development. Indeed, it was shown to be involved in DNA G-quadruplex biology and RNA metabolism, most importantly in the miRNA maturation pathway and the decay of oxidized- or abasic-miRNAs during oxidative stress conditions. Furthermore, in recent years several non-canonical pathways of miRNA biogenesis have been described, with a specific focus on guanosine-rich precursors that can form RNA G-quadruplex (rG4) structures. In this study, we show that several miRNA precursors, dysregulated upon APE1-depletion, contain an rG4 motif and that their corresponding target genes are upregulated after APE1-depletion. We also show, both by in vitro assays and by using a HeLa cell model, that APE1 can bind and regulate the folding of an rG4 structure contained in pre-miR92b, with a mechanism strictly dependent on critical lysine residues present in the N-terminal disordered region. Furthermore, APE1 depletion in HeLa cells alters the maturation process of miR-92b, mainly affecting the shuttling between the nucleus and cytosol. Lastly, bioinformatic analysis of APE1-regulated rG4-containing miRNAs supports the relevance of our findings for cancer biology. Specifically, these miRNAs exhibit high prognostic significance in lung, cervical, and liver cancer, as suggested by their involvement in several cancer-related pathways.Significance Statement We highlight an undescribed non-canonical role of the mammalian Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APE1) in the context of RNA G-quadruplexes (rG4), specifically in the alternative pathway of miRNA maturation of guanosine-rich miRNA precursors. Specifically, APE1 binds these structures and modulates their folding, mainly through its N-terminal region and some residues in its catalytic domain. Moreover, we showed an interesting new role of APE1 in regulating the shuttling and accumulation of miR-92b between the nuclear and cytosolic compartments, opening new perspectives on how APE1 may exercise its role in the miRNA maturation pathway and function. Moreover, APE1-depleted dysregulated miRNAs with rG4 motifs in their precursors have significant prognostic value in lung, cervical, and liver tumors, suggesting potential targets for cancer therapy.### Competing Interest StatementThe authors have declared no competing interest.

Sequencing for RNA modifications with the nanopore direct RNA sequencing platform provides ionic current levels, helicase dwell times, and base call data that differentiate the modifications from the canonical form. Herein, model RNAs were synthesized with site-specific uridine (U) base modifications that enable the study of increasing an alkyl group size, halogen identity, or a change in base acidity to impact the nanopore data. The analysis concluded that increases in alkyl size trend with greater current blockage but a similar change in base-call error was not found. The addition of a halogen series to C5 of U revealed that the current levels recorded a trend with the water-octanol partition coefficient of the base, as well as the base call error. Studies with U modifications that are deprotonated (i.e., anionic) under the sequencing conditions gave broad current levels that influenced the base call error. Some modifications led to helicase dwell time changes. These insights provide design parameters for modification-specific chemical reagents that can shift nanopore signatures to minimize false positive reads, a known issue with this sequencing approach.

Molecular details for the impact of DNA damage on folding of potential G-quadruplex sequences (PQSs) to noncanonical DNA structures involved in gene regulation are poorly understood. Here, the effects of DNA base damage and strand breaks on PQS folding kinetics were studied in the context of the VEGF promoter sequence embedded between two DNA duplex anchors, termed a duplex-G-quadruplex-duplex (DGD) motif. This DGD scaffold imposes constraints on the PQS folding process that more closely mimic those found in genomic DNA. Folding kinetics were monitored by circular dichroism (CD) to find folding half-lives ranging from 2 s to 12 min depending on the DNA damage type and sequence position. The presence of Mg2+ ions and G-quadruplex (G4)-binding protein APE1 facilitated the folding reactions. A strand break placing all four G runs required for G4 formation on one side of the break accelerated the folding rate by >150-fold compared to the undamaged sequence. Combined 1D 1H NMR and CD analyses confirmed that isothermal folding of the VEGF-DGD constructs yielded spectral signatures that suggest the formation of G4 motifs and demonstrated a folding dependency on the nature and location of DNA damage. Importantly, the PQS folding half-lives measured are relevant to replication, transcription, and DNA repair time frames.

The DNA repair endonuclease APE1 is responsible for the cleavage of abasic sites (AP) in DNA as well as binding AP in promoter G-quadruplex (G4) folds in some genes to regulate transcription. The present studies focused on the topological properties of AP-bearing G4 folds and how they impact APE1 interaction. The human telomere sequence with a tetrahydrofuran model (F) of an AP was folded in K+- or Na+-containing buffers to adopt hybrid- or basket-folds, respectively. Endonuclease and binding assays were performed with APE1 and the G4 substrates, and the data were compared to prior work with parallel-stranded VEGF and NEIL3 promoter G4s to identify topological differences. The APE1-catalyzed endonuclease assays led to the conclusion that telomere G4 folds were slightly better substrates than the promoter G4s, but the yields were all low compared to duplex DNA. In the binding assays, G4 topological differences were observed in which APE1 bound telomere G4s with dissociation constants similar to single-stranded DNA, and promoter G4s were bound with nearly ten-fold lower values similar to duplex DNA. An in-cellulo assay with the telomere G4 in a model promoter bearing a lesion failed to regulate transcription. These data support a hypothesis that G4 topology in gene promoters is a critical feature that APE1 recognizes for gene regulation.

The bacterium Escherichia coli possesses 16S and 23S rRNA strands that have 36 chemical modification sites with 17 different structures. Nanopore direct RNA sequencing using a protein nanopore sensor and helicase brake, which is also a sensor, was applied to the rRNAs. Nanopore current levels, base calling profile, and helicase dwell times for the modifications relative to unmodified synthetic rRNA controls found signatures for nearly all modifications. Signatures for clustered modifications were determined by selective sequencing of writer knockout E. coli and sequencing of synthetic RNAs utilizing some custom-synthesized nucleotide triphosphates for their preparation. The knowledge of each modification’s signature, apart from 5-methylcytidine, was used to determine how metabolic and cold-shock stress impact rRNA modifications. Metabolic stress resulted in either no change or a decrease, and one site increased in modification occupancy, while cold-shock stress led to either no change or a decrease. The double modification m4Cm1402 resides in 16S rRNA, and it decreased with both stressors. Using the helicase dwell time, it was determined that the N 4 methyl group is lost during both stressors, and the 2′-OMe group remained. In the ribosome, this modification stabilizes binding to the mRNA codon at the P-site resulting in increased translational fidelity that is lost during stress. The E. coli genome has seven rRNA operons (rrn), and the earlier studies aligned the nanopore reads to a single operon (rrnA). Here, the reads were aligned to all seven operons to identify operon-specific changes in the 11 pseudouridines. This study demonstrates that direct sequencing for >16 different RNA modifications in a strand is achievable.

Oxidative damage to DNA nucleotides has many cellular outcomes that could be aided by the development of sequencing methods. Herein, the previously reported click-code-seq method for sequencing a single damage type is redeveloped to enable the sequencing of many damage types by making simple changes to the protocol (i.e., click-code-seq v2.0).

Direct RNA sequencing with a commercial nanopore platform was used to sequence RNA containing uridine (U), pseudouridine (Ψ), or N1-methylpseudouridine (m1Ψ) generated by in vitro transcription (IVT). The base calling data as well as the ionic currents and dwell times for U, Ψ, or m1Ψ as they translocated through the helicase and nanopore proteins identified diagnostic signatures for Ψ and m1Ψ; however, the two modifications yielded similar patterns although both were different from U. Understanding the nanopore signatures for Ψ and m1Ψ enabled a running start T7 RNA polymerase assay to study how competing mixtures of UTP with ΨTP or m1ΨTP lead to nucleotide selection in all possible adjacent sequence contexts. For UTP vs. ΨTP, ΨTP was favorably incorporated in singly-modified contexts, while doubly-modified contexts found high yields of ΨTP insertion on the 5′ side and lower yields on the 3′ side. For UTP vs. m1ΨTP, UTP was favorably selected except in 5′-XA (X = U or m1Ψ) where the ratio was determined by their relative NTP concentrations. Experiments with chemically-modified triphosphates and DNA templates designed based on the structure of T7 RNA polymerase provide a model to explain the observations. These results may aid in future efforts that employ IVT to make therapeutic mRNAs with sub-stochiometric amounts of m1Ψ.

Nanopore direct RNA sequencing is a technology that allows sequencing for epitranscriptomic modifications with the possibility of a quantitative assessment. In the present work, pseudouridine (psi) was sequenced with the nanopore before and after the pH 7 bisulfite reaction that yields stable ribose adducts at C1 ' of psi. The adducted sites produced greater base call errors in the form of deletion signatures compared to psi. Sequencing studies on E. coli rRNA and tmRNA before and after the pH 7 bisulfite reaction demonstrated that using chemically-assisted nanopore sequencing has distinct advantages for minimization of false positives and false negatives in the data. The rRNA from E. coli has 19 known U/C sequence variations that give similar base call signatures as psi, and therefore, are false positives when inspecting base call data; however, these sites are refractory to reacting with bisulfite as is easily observed in nanopore data. The E. coli tmRNA has a low occupancy psi in a pyrimidine-rich sequence context that is called a U representing a false negative; partial occupancy by psi is revealed after the bisulfite reaction. In a final study, 5-methylcytidine (m5C) in RNA can readily be observed after the pH 5 bisulfite reaction in which the parent C deaminates to U and the modified site does not react. This locates m5C when using bisulfite-assisted nanopore direct RNA sequencing, which is otherwise challenging to observe. The advantages and challenges of the overall approach are discussed. Nanopore direct RNA sequencing assisted by pseudouridine- and m5C-specific bisulfite treatment is a technology that allows sequencing for epitranscriptomic modifications with the possibility of quantitative assessment.