Search

Fluorescent dyes are routinely used to visualize DNA or RNA in various experiments, and some dyes also act as photosensitizers capable of catalyzing oxidation reactions. The present studies explored whether the common labeling dyes fluorescein, rhodamine, BODIPY, or cyanine3 (Cy3) can function as photosensitizers to oxidize nucleic acid polymers. Photoirradiation of each dye in the presence of the guanine (G) heterocycle, which is the most sensitive toward oxidation, identified slow rates of nucleobase oxidation in the nucleoside and DNA contexts. For all four fluorophores studied, the only product detected was spiroiminodihydantoin (Sp) suggesting the dyes functioned as Type II photosensitizers and generate singlet oxygen (

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.

APE1 complexed with an abasic site in G-quadruplex (G4) topologies displays differences in binding constants, enzyme cleavage yields, and gene regulation. APE1 tightly binds parallel G4s to regulate transcription that is not found with hybrid G4s.

The binding interaction between the DNA repair enzyme apurinic/apyrimidinic endonuclease-1 (APE1) with promoter G-quadruplex (G4) folds bearing an abasic site (AP) can serve as a gene regulatory switch during oxidative stress. Prior fluorescence-based analysis in solution suggested APE1 binds the

Exposure of DNA to oxidants results in modification of the electron-rich guanine heterocycle including formation of the mutagenic 5-carboxamido-5-formamido-2-iminohydantoin (2Ih) lesion. Previously thought to exist solely as a pair of diastereomers, we found under biologically relevant conditions that 2Ih reversibly closes to a formerly hypothetical intermediate and opens into a newly discovered regioisomer. In a nucleoside model, only ∼80% of 2Ih existed as the structure studied over the last 20 years with significant isomeric products persisting in buffered aqueous solution.

In RNA, pseudouridine (

The G-quadruplex is a noncanonical fold of DNA commonly found at telomeres and within gene promoter regions of the genome. These guanine-rich sequences are highly susceptible to damages such as base oxidation and depurination, leading to abasic sites. In the present work, we address whether a vacancy, such as an abasic site, in a G-quadruplex serves as a specific ligand recognition site. When the G-tetrad is all guanines, the vacant (abasic) site is recognized and bound by free guanine nucleobase. However, we aim to understand whether the preference for a specific ligand recognition changes with the presence of a guanine oxidation product 8-oxo-7,8-dihydroguanine (OG) adjacent to the vacancy in the tetrad. Using molecular dynamics simulation, circular dichroism, and nuclear magnetic resonance, we examined the ability for riboflavin to stabilize abasic site-containing G-quadruplex structures. Through structural and free energy binding analysis, we observe riboflavin's ability to stabilize an abasic site-containing G-quadruplex only in the presence of an adjacent OG-modified base. Further, when compared to simulation with the vacancy filled by free guanine, we observe that the free guanine nucleobase is pushed outside of the tetrad by OG to interact with other parts of the structure, including loop residues. These results support the preference of riboflavin over free guanine to fill an OG-adjacent G-quadruplex abasic vacancy.

Escherichia colipossess the 16S and 23S rRNA strands that have 36 chemical modification sites with 17 different structures. Direct RNA nanopore 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 non-modified synthetic rRNA controls found signatures for nearly all modifications. Signatures for clustered modifications were determined by selective sequencing of writer knock-outE. coliand 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. In the 16S rRNA, there resides an m4Cmmodification at site 1402 that decreased with both stressors. Using helicase dwell time, it was determined that theN4methyl 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. TheE. coligenome has seven rRNA operons (rrn), and earlier studies aligned the nanopore reads to a single operon (rrnA). Here, the reads were aligned to the 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.

Purpose One outcome of DNA damage from hydroxyl radical generated by ionizing radiation (IR) or by the Fenton reaction is oxidation of the nucleobases, especially guanine (G). While 8-oxo-7,8-dihydroguanine (OG) is a commonly studied oxidized lesion, several others are formed in high abundance, including 5-carboxamido-5-formamido-2-iminohydantoin (2Ih), a prevalent product in in vitro chemistry that is challenging to study from cellular sources. In this short review, we have a goal of explaining new insights into hydroxyl radical-induced oxidation chemistry of G in DNA and comparing it to endogenous DNA damage, as well as commenting on the biological outcomes of DNA base damage. Conclusions Pathways of oxidation of G are discussed and a comparison is made between IR (hydroxyl radical chemistry) and endogenous oxidative stress that largely forms carbonate radical anion as a reactive intermediate. These pathways overlap with the formation of OG and 2Ih, but other guanine-derived lesions are more pathway specific. The biological consequences of guanine oxidation include both mutagenesis and epigenetics; a new mechanism of gene regulation via the base excision repair pathway is described for OG, whereas the impact of IR in forming guanine modifications may be to confound this process in addition to introduction of mutagenic sites.

The base excision repair enzyme apurinic/apyrimidinic endonuclease-1 (APE1) is also engaged in transcriptional regulation. APE1 can function in both pathways when the protein binds to a promoter G-...

Oxidation of guanine generates several types of DNA lesions, such as 8-oxoguanine (8OG), 5-guanidinohydantoin (Gh), and spiroiminodihydantoin (Sp). These guanine-derived oxidative DNA lesions interfere with both replication and transcription. However, the molecular mechanism of transcription processing of Gh and Sp remains unknown. In this study, by combining biochemical and structural analysis, we revealed distinct transcriptional processing of these chemically related oxidized lesions: 8OG allows both error-free and error-prone bypass, whereas Gh or Sp causes strong stalling and only allows slow error-prone incorporation of purines. Our structural studies provide snapshots of how polymerase II (Pol II) is stalled by a nonbulky Gh lesion in a stepwise manner, including the initial lesion encounter, ATP binding, ATP incorporation, jammed translocation, and arrested states. We show that while Gh can form hydrogen bonds with adenosine monophosphate (AMP) during incorporation, this base pair hydrogen bonding is not sufficient to hold an ATP substrate in the addition site and is not stable during Pol II translocation after the chemistry step. Intriguingly, we reveal a unique structural reconfiguration of the Gh lesion in which the hydantoin ring rotates ∼90° and is perpendicular to the upstream base pair planes. The perpendicular hydantoin ring of Gh is stabilized by noncanonical lone pair–π and CH–π interactions, as well as hydrogen bonds. As a result, the Gh lesion, as a functional mimic of a 1,2-intrastrand crosslink, occupies canonical −1 and +1 template positions and compromises the loading of the downstream template base. Furthermore, we suggest Gh and Sp lesions are potential targets of transcription-coupled repair.

Contrary to frequent reports in the literature, hydroxyl radical is not a key species participating in endogenous oxidative DNA damage. Instead, carbonate radical anion is formed from the Fenton reaction under cellular conditions and from decomposition of nitrosoperoxycarbonate generated during inflammation. Carbonate radical anion is a potent one-electron oxidant capable of generating base radical cations that can migrate over long distances in duplex DNA, ultimately generating 8-oxo-7,8-dihydroguanine at a redox-sensitive sequence such as GGG. Such a mechanism enables G-quadruplex-forming sequences to act as long-range sensors of oxidative stress, impacting gene expression via the DNA repair mechanism that reads and ultimately erases the oxidized base. With a writing, reading and erasing mechanism in place, oxidative ‘damage’ to DNA might be relabeled as ‘epigenetic’ modifications.

The base excision repair enzyme apurinic/apyrimidinic endonuclease-1 (APE1) is also engaged in transcriptional regulation. APE1 can function in both pathways when the protein binds to a promoter G-quadruplex (G4) bearing an abasic site (modeled with tetrahydrofuran, F) that leads to enzymatic stalling on the non-canonical fold to recruit activating transcription factors. Biochemical and biophysical studies to address APE1’s binding and catalytic activity with the vascular endothelial growth factor (VEGF) promoter G4 are lacking, and the present work provides insight on this topic. Herein, the native APE1 was used for cleavage assays, and the catalytically inactive mutant D210A was used for binding assays with double-stranded DNA (dsDNA) versus the native G4 or the G4 with F at various positions, revealing dependencies of the interaction on the cation concentrations K(+) and Mg(2+) and the N-terminal domain of the protein. Assays in 0, 1, or 10 mM Mg(2+) found that dsDNA and G4 substrates required the cation for both binding and catalysis, in which G4 binding increased with [Mg(2+)]. Studies with 50 versus physiological 140 mM K(+) ions showed that F-containing dsDNA was bound and cleaved by APE1; whereas, the G4s with F were poorly cleaved in low salt and not cleaved at all at higher salt while the binding remained robust. Using Δ33 or Δ61 N-terminal truncated APE1 proteins, the binding and cleavage of dsDNA with F was minimally impacted; in contrast, the G4s required the N-terminus for binding and catalysis is nearly abolished without the N-terminus. With this knowledge, we found APE1 could remodel the F-containing VEGF promoter dsDNA→G4 folds in solution. Lastly, the addition of the G4 ligand pyridostatin inhibited APE1 binding and cleavage of F-containing G4s but not dsDNA. The biological and medicinal chemistry implications of the results are discussed.

Nucleic acids are chemically modified to fine-tune their properties for biological function. Chemical tools for selective tagging of base modifications enables new approaches; the photosensitizers riboflavin and anthraquinone were previously proposed to oxidize N(6)-methyladenine (m(6)A) or 5-methylcytosine (5mdC) selectively. Herein, riboflavin, anthraquinone, or Rose Bengal were allowed to react with the canonical nucleosides dA, dC, dG, and dT, and the modified bases 5mdC, m(6)A, 8-oxoguanine (dOG), and 8-oxoadenine (dOA) to rank their reactivities. The nucleoside studies reveal that dOG is the most reactive and that the native nucleoside dG is higher or similar in reactivity to 5mdC or m(6)A; competition in both single- and double-stranded DNA of dG vs. 5mdC or 6mdA for oxidant confirmed that dG is favorably oxidized. Thus, photosensitizers are promiscuous nucleic acid oxidants with poor chemoselectivity that will negatively impact attempts at targeted oxidation of modified nucleotides in cells.

The base excision repair enzyme apurinic/apyrimidinic endonuclease-1 (APE1) is also engaged in transcriptional regulation. APE1 can function in both pathways when the protein binds to a promoter G-quadruplex (G4) bearing an abasic site (modeled with tetrahydrofuran, F) that leads to enzymatic stalling on the non-canonical fold to recruit activating transcription factors. Biochemical and biophysical studies to address APE1’s binding and catalytic activity with the vascular endothelial growth factor (VEGF) promoter G4 are lacking, and the present work provides insight on this topic. Herein, the native APE1 was used for cleavage assays, and the catalytically inactive mutant D210A was used for binding assays with double-stranded DNA (dsDNA) versus the native G4 or the G4 with F at various positions, revealing dependencies of the interaction on the cation concentrations K+and Mg2+and the N-terminal domain of the protein. Assays in 0, 1, or 10 mM Mg2+found dsDNA and G4 substrates required the cation for both binding and catalysis, in which G4 binding increased with [Mg2+]. Studies with 50 versus physiological 140 mM K+ions present showed that F-containing dsDNA was bound and cleaved by APE1; whereas, the G4s with F were poorly cleaved in low salt and not cleaved at all at higher salt while the binding remained robust. Using Δ33 or Δ61 N-terminal truncated APE1 proteins, the binding and cleavage of dsDNA with F was minimally impacted; in contrast, the G4s required the N-terminus for binding and catalysis. With this knowledge, we found APE1 could remodel the F-containingVEGFpromoter dsDNA→G4 folds in solution. Lastly, the addition of the G4 ligand pyridostatin inhibited APE1 binding and cleavage of F-containing G4s but not dsDNA. The biological and medicinal chemistry implications of the results are discussed.TOC graphic

Many cancer-associated genes are regulated by guanine (G)-rich sequences that are capable of refolding from the canonical duplex structure to an intrastrand G-quadruplex. These same sequences are sensitive to oxidative damage that is repaired by the base excision repair glycosylases OGG1 and NEIL1–3. We describe studies indicating that oxidation of a guanosine base in a gene promoter G-quadruplex can lead to up- and downregulation of gene expression that is location dependent and involves the base excision repair pathway in which the first intermediate, an apurinic (AP) site, plays a key role mediated by AP endonuclease 1 (APE1/REF1). The nuclease activity of APE1 is paused at a G-quadruplex, while the REF1 capacity of this protein engages activating transcription factors such as HIF-1α, AP-1 and p53. The mechanism has been probed by in vitro biophysical studies, whole-genome approaches and reporter plasmids in cellulo. Replacement of promoter elements by a G-quadruplex sequence usually led to upregulation, but depending on the strand and precise location, examples of downregulation were also found. The impact of oxidative stress-mediated lesions in the G-rich sequence enhanced the effect, whether it was positive or negative., Graphical Abstract Graphical AbstractGuanine-rich sequences in promoter regions of genes are sensitive to oxidative stress and may refold to G-quadruplexes. The DNA repair process impacts gene expression through the ability of AP-endonuclease to bind and recruit activating transcription factors.

Living in an oxygen atmosphere demands an ability to thrive in the presence of reactive oxygen species (ROS). Aerobic organisms have successfully found solutions to the oxidative threats imposed by ROS by evolving an elaborate detoxification system, upregulating ROS during inflammation, and utilizing ROS as messenger molecules. In this Perspective, recent studies are discussed that demonstrate ROS as signaling molecules for gene regulation by combining two emergent properties of the guanine (G) heterocycle in DNA, namely, oxidation sensitivity and a propensity for G-quadruplex (G4) folding, both of which depend upon sequence context. In human gene promoters, this results from an elevated 5'-GG-3' dinucleotide frequency and GC enrichment near transcription start sites. Oxidation of DNA by ROS drives conversion of G to 8-oxo-7,8-dihydroguanine (OG) to mark target promoters for base excision repair initiated by OG-glycosylase I (OGG1). Sequence-dependent mechanisms for gene activation are available to OGG1 to induce transcription. Either OGG1 releases OG to yield an abasic site driving formation of a non-canonical fold, such as a G4, to be displayed to apurinic/apyrimidinic 1 (APE1) and stalling on the fold to recruit activating factors, or OGG1 binds OG and facilitates activator protein recruitment. The mechanisms described drive induction of stress response, DNA repair, or estrogen-induced genes, and these pathways are novel potential anticancer targets for therapeutic intervention. Chemical concepts provide a framework to discuss the regulatory or possible epigenetic potential of the OG modification in DNA, in which DNA "damage" and non-canonical folds collaborate to turn on or off gene expression. The next steps for scientific discovery in this growing field are discussed.

The recent report of RBS-Seq to map simultaneously the epitranscriptomic modifications N1-methyladenosine, 5-methylcytosine, and pseudouridine (Ψ) via bisulfite treatment of RNA provides a key advance to locate these important modifications. The locations of Ψ were found by a deletion signature generated during cDNA synthesis after bisulfite treatment for which the chemical details of the reaction are poorly understood. In the present work, the bisulfite reaction with Ψ was explored to identify six isomers of bisulfite adducted to Ψ. We found four of these adducts involved the heterocyclic ring, similar to the reaction with other pyrimidines. The remaining two adducts were bonded to the 1' carbon, which resulted in opening of the ribose ring. The utilization of complementary 1D- and 2D-NMR, Raman, and electronic circular dichroism spectroscopies led to the assignment of the two ribose adducts being the constitutional isomers of an S- and an O-adduct of bisulfite to the ribose, and these are the final products after heating. A mechanistic proposal is provided to rationalize chemically the formation and stereochemistries of all six isomeric bisulfite adducts to Ψ; conversion of intermediate adducts to the two final products is proposed to involve E2, SN2', and [2,3]-sigmatropic shift reactions. Lastly, a synthetic RNA template with Ψ at a known location was treated with bisulfite, leading to a deletion signature after reverse transcription, supporting the RBS-Seq report. This classical bisulfite reaction used for epigenomic and epitranscriptomic sequencing diverges from the C nucleoside Ψ to form stable bisulfite end products that yield signatures for next-generation sequencing.

The NEIL3 DNA repair gene is induced in cells or animal models experiencing oxidative or inflammatory stress along with oxidation of guanine (G) to 8-oxo-7,8-dihydroguanine (OG) in the genome. We hypothesize that a G-rich promoter element that is a potential G-quadruplex-forming sequence (PQS) in NEIL3 is a site for introduction of OG with epigenetic-like potential for gene regulation. Activation occurs when OG is formed in the NEIL3 PQS located near the transcription start site. Oxidative stress either introduced by TNFα or synthetically incorporated into precise locations focuses the base excision repair process to read and catalyze removal of OG via OG-glycosylase I (OGG1), yielding an abasic site (AP). Thermodynamic studies showed that AP destabilizes the duplex, enabling a structural transition of the sequence to a G-quadruplex (G4) fold that positions the AP in a loop facilitated by the NEIL3 PQS having five G runs in which the four unmodified runs adopt a stable G4. This presents AP to apurinic/apyrimidinic endonuclease 1 (APE1) that poorly cleaves the AP backbone in this context according to in vitro studies, allowing the protein to function as a trans activator of transcription. The proposal is supported by chemical studies in cellulo and in vitro. Activation of NEIL3 expression via the proposed mechanism allows cells to respond to mutagenic DNA damage removed by NEIL3 associated with oxidative or inflammatory stress. Lastly, inspection of many mammalian genomes identified conservation of the NEIL3 PQS, suggesting this sequence was favorably selected to function as a redox switch with OG as the epigenetic-like regulatory modification.

Oxidative damage to DNA can lead to DNA-protein cross-links which can interfere with DNA transcription, replication, and repair. In experimental studies modeling oxidative damage to DNA, oxidation of guanosine by sulfate radical anion in the presence of lysine produced a mixture of lysine (Lys)-substituted spiroiminodihydantoins (Sp): ∼65% 5-Lys-Sp, ∼30% 8-Lys-Sp, and ∼5% 5,8-diLys-Sp. Pathways for formation of the lysine adducts during the oxidation of guanine by sulfate radical anions have been mapped out using B3LYP density functional theory and the SMD solvation model. Methylamine was used as a model for lysine, and imidazole served as a proton acceptor. The lowest barrier for methylamine reaction with guanine radical is addition at C8, yielding mainly 8-NHR-Sp and some 5,8-diNR-Sp. This is in good agreement with the cross-link ratios for mild oxidations mediated by type I photosensitizers such as benzophenone, but this is not in agreement with the product ratios for strong oxidants such as sulfate radical anion. The calculations explored pathways for oxidation of guanine by sulfate radical anion that produced guanine radical and radical cation and doubly oxidized guanine (Gox) and its cation. Sulfate radical anion can also oxidize methylamine to produce neutral methylamine radical (CH3NH•) after deprotonation. The calculations qualitatively reproduced the observed product ratio at pH 7 via a pathway involving the barrierless addition of methylamine radical at C5 and C8 of guanine radical. After C5 addition of methylamine radical, the lowest barrier is for H2O addition at C8 leading exclusively to 5-NHR-Sp. After C8 addition of methylamine radical, H2O and methylamine addition to C5 lead to 8-NHR-Sp and some 5,8-diNR-Sp.

Significance The field of RNA modification would be significantly advanced by the development of sensitive, accurate, single-base resolution methods for profiling multiple common RNA modifications in the same RNA molecule. Our work provides several advances toward that goal, including (i) quantitative methods for profiling Ψ sites at true base-pair resolution transcriptome-wide, (ii) a chemical understanding of our observed Ψ-dependent deletion signature, (iii) improved methods for profiling m5C and m1A, and (iv) a coupling of these methods for the simultaneous detection of all three modifications in the same RNA. Together, the combinatorial ability and relative ease of execution provided by this procedure should greatly forward epitranscriptome studies involving these three very common RNA modifications., The breadth and importance of RNA modifications are growing rapidly as modified ribonucleotides can impact the sequence, structure, function, stability, and fate of RNAs and their interactions with other molecules. Therefore, knowing cellular RNA modifications at single-base resolution could provide important information regarding cell status and fate. A current major limitation is the lack of methods that allow the reproducible profiling of multiple modifications simultaneously, transcriptome-wide and at single-base resolution. Here we developed RBS-Seq, a modification of RNA bisulfite sequencing that enables the sensitive and simultaneous detection of m5C, Ψ, and m1A at single-base resolution transcriptome-wide. With RBS-Seq, m5C and m1A are accurately detected based on known signature base mismatches and are detected here simultaneously along with Ψ sites that show a 1–2 base deletion. Structural analyses revealed the mechanism underlying the deletion signature, which involves Ψ-monobisulfite adduction, heat-induced ribose ring opening, and Mg2+-assisted reorientation, causing base-skipping during cDNA synthesis. Detection of each of these modifications through a unique chemistry allows high-precision mapping of all three modifications within the same RNA molecule, enabling covariation studies. Application of RBS-Seq on HeLa RNA revealed almost all known m5C, m1A, and ψ sites in tRNAs and rRNAs and provided hundreds of new m5C and Ψ sites in noncoding RNAs and mRNAs. However, our results diverge greatly from earlier work, suggesting ∼10-fold fewer m5C sites in noncoding and coding RNAs and the absence of substantial m1A in mRNAs. Taken together, the approaches and refined datasets in this work will greatly enable future epitranscriptome studies.

One response to oxidation of guanine (G) to 8-oxo-7,8-dihydroguanine (OG) in a gene promoter is regulation of mRNA expression suggesting an epigenetic-like role for OG. A proposed mechanism involves G oxidation within a potential G-quadruplex-forming sequence (PQS) in the promoter enabling a structural shift from B-DNA to a G-quadruplex fold (G4). When OG was located in the coding vs. template strand, base excision repair led to an on/off transcriptional switch. Herein, a G-rich, potential Z-DNA-forming sequence (PZS) comprised of a d(GC)(n) repeat was explored to determine whether oxidation in this motif was also a transcriptional switch. Bioinformatic analysis found 1650 PZSs of length >10 nts in the human genome that were overrepresented in promoters and 5′-UTRs. Studies in human cells transfected with a luciferase reporter plasmid in which OG was synthesized in a PZS context in the promoter found that a coding strand OG increased expression, and a template strand OG decreased expression. The initial base excision repair product of OG, an abasic site (AP), was also found to yield similar expression changes as OG. Biophysical studies on model Z-DNA strands found OG favored a shift in the equilibrium to Z-DNA from B-DNA, while an AP disrupted Z-DNA to favor a hairpin placing AP in the loop where it is a poor substrate for the endonuclease APE1. Overall, the impact of OG and AP in a PZS on gene expression was similar to that in a PQS but reduced in magnitude.

Oxidation of the guanine (G) heterocycle to 8-oxo-7,8-dihydroguanine (OG) in mammalian gene promoters was demonstrated to induce transcription. Potential G-quadruplex forming sequences (PQSs) in promoters have a high density of G nucleotides rendering them highly susceptible to oxidation and possible gene activation. The VEGF PQS with OG or an abasic site were synthesized at key locations in the SV40 or HSV-TK model promoters to determine the location dependency in the gene expression profile in human cells. The PQS location with respect to the transcription start site (TSS) and strand of occupancy (coding versus non-coding strand) are key parameters that determine the magnitude and direction in which gene expression changes with the chemically modified VEGF PQS. The greatest impact observed for OG or F in the PQS context in these promoters was within ∼200 bp of the TSS. Established PQSs found to occur naturally in a similar location relative to the TSS for possible oxidation-induced gene activation include c-MYC, KRAS, c-KIT, HIF-1α, PDGF-A and hTERT. The studies provide experimental constraints that were used to probe bioinformatic data regarding PQSs in the human genome for those that have the possibility to be redox switches for gene regulation.

Because of its low redox potential, guanine (G) is the most frequent site of oxidation in the genome. Metabolic processes generate reactive oxygen species (ROS) that can oxidize G to yield 8-oxo-7,8-dihydroguanine (OG) as a key two-electron oxidation product. In a genome, G-rich sites including many gene promoters are sensitive to oxidative modification, and some of these regions have the propensity to form G-quadruplexes (G4s). Recently, OG formation in G-rich gene promoters was demonstrated to regulate mRNA expression via the base excision repair (BER) pathway. The proliferating cell nuclear antigen ( PCNA) gene was previously found to be activated by metabolic ROS, and the gene has a five G-track potential G4 in the coding strand of its promoter. Herein, we demonstrated the ability for four G runs of the PCNA promoter sequence to adopt a parallel-stranded G4. Next, we identified G nucleotides in the PCNA G4 sequence sensitive to oxidative modification. The G oxidation product OG and its initial BER product, an abasic site, were synthetically incorporated into the four- and five-track PCNA sequences at the sensitive sites followed by interrogation of G4 folding by five methods. We found the modifications impacted the G4 folds with positional dependency. Additionally, the fifth G track maintained the stability of the modified G4s by extrusion of the oxidatively modified G run. Finally, we synthetically inserted a portion of the promoter into a reporter plasmid with OG at select oxidation-prone positions to monitor expression in human glioblastoma cells. Our results demonstrate that OG formation in the context of the PCNA G4 can lead to increased gene expression consistent with the previous studies identifying that metabolic ROS activates transcription of the gene. This study provides another example of a G4 with the potential to serve as a regulatory agent for gene expression upon G oxidation.

Nanopore devices can directly sequence RNA, and the method has the potential to determine locations of epitranscriptomic modifications that have grown in significance because of their roles in cell regulation and stress response. Pseudouridine (Ψ), the most common modification in RNA, was sequenced with a nanopore system using a protein sensor with a helicase brake in synthetic RNAs with 100% modification at 18 known human pseudouridinylation sites. The new signals were compared to native uridine (U) control strands to characterize base calling and associated errors as well as ion current and dwell time changes. The data point to strong sequence context effects in which Ψ can easily be detected in some contexts while in others Ψ yields signals similar to U that would be false negatives in an unknown sample. We identified that the passage of Ψ through the helicase brake slowed the translocation kinetics compared to U and showed a smaller sequence bias that could permit detection of this modification in RNA. The unique signals from Ψ relative to U are proposed to reflect the syn-anti conformational flexibility of Ψ not found in U, and the difference in π stacking between these bases. This observation permitted analysis of SARS-CoV-2 nanopore sequencing data to identify five conserved Ψ sites on the 3’ end of the viral sub-genomic RNAs, and other less conserved Ψ sites. Using the helicase as a sensor protein in nanopore sequencing experiments enables detection of this modification in a greater number of relevant sequence contexts. The data are discussed concerning their analytical and biological significance.

Sequences of DNA typically adopt B-form duplexes in genomes, although noncanonical structures such as G-quadruplexes, i-motifs, Z-DNA, and cruciform structures can occur. A challenge is to determine the functions of these various structures in cellular processes. We and others have hypothesized that G-rich G-quadruplex-forming sequences in human genome promoters serve to sense oxidative damage generated during oxidative stress impacting gene regulation. Herein, chemical tools and a cell-based assay were used to study the oxidation of guanine to 8-oxo-7,8-dihydroguanine (OG) in the context of a cruciform-forming sequence in a gene promoter to determine the impact on transcription. We found that OG in the nontemplate strand in the loop of a cruciform-forming sequence could induce gene expression; conversely when OG was in the same sequence on the template strand, gene expression was inhibited. A model for the transcriptional changes observed is proposed in which OG focuses the DNA repair process on the promoter to impact expression. Our cellular and biophysical studies and literature sources support the idea that removal of OG from duplex DNA by OGG1 yields an abasic site (AP) that triggers a structural shift to the cruciform fold. The AP-bearing cruciform structure is presented to APE1, which functions as a conduit between DNA repair and gene regulation. The significance is enhanced by a bioinformatic study of all human gene promoters and transcription termination sites for inverted repeats (IRs). Comparison of the two regions showed that promoters have stable and G-rich IRs at a low frequency and termination sites have many AT-rich IRs with low stability.

The following joint Editorial was originally published in ACS Applied Materials & Interfaces (DOI: 10.1021/acsami.0c10979). We confront the terrible reality that systemic racism and discrimination impacts the daily personal and professional lives of many members of the scientific community and broader society. In the U.S., the brutal killing of George Floyd while in police custody is one of the most recent examples of the centuries of systemic violence suffered by Black Americans. This moment and its aftermath lay bare the legacies of racism and its exclusionary practices. Let us be clear: we, the Editors, Staff, and Governance Members of ACS Publications condemn the tragic deaths of Black people and stand in solidarity with Black members of the science and engineering community. Moreover, ACS condemns racism, discrimination, and harassment in all forms. We will not tolerate practices and viewpoints that exclude or demean any member of our community. Despite these good intentions, we recognize that our community has not done enough to provide an environment for Black chemists to thrive. Rep. Eddie Bernice Johnson, Chairwoman of the U.S. House Committee on Science, Space, and Technology said, “So far, we have gotten by with a STEM workforce that does not come close to representing the diversity of our nation. However, if we continue to leave behind so much of our nation’s brainpower, we cannot succeed.”(1) Indeed, the U.S. National Science Foundation notes that Blacks and other under-represented minority groups continue to be under-represented in science and engineering education and employment.(2) What is abundantly clear in this moment is that this lack of representation is a symptom of systemic racism across all levels of education and professional life. We know that supportive words are not enough. We must develop and implement a concrete plan for changing our trajectory. Publications and citations are academic currency, and while we like to think publishing a manuscript is “just about the science”, we know that is not true for everyone. We have seen the biases (largely through the lens of gender and in Western countries because of the limitations in bibliometric analyses) and applaud our colleagues at the RSC for their massive study that explored these gender barriers in the publishing pipeline(3) and their recent Inclusion and Diversity Framework.(4) At the present time, unfortunately, less is known about the effects of race and ethnicity on publishing success. A study published in PeerJ, however, found that unprofessional reviewer comments had a disproportionate effect on authors from under-represented groups.(5) As the world’s leading society publisher, we have a responsibility to aggressively combat bias in all aspects of the publishing process, including systemic under-representation of Blacks in this endeavor (no ACS journal is currently led by a Black Editor-in-Chief). Within ACS Publications, we actively track gender and geographic diversity of editors, advisors, authors, and reviewers, and we anecdotally report on race of editors. Diversity encompasses many more dimensions than these, and we acknowledge that we can do much more than we have. We affirm that diversity and inclusion strengthen the research community and its impact, and we are committed to developing, implementing, tracking, and reporting on our progress to ensure that our editors, advisors, reviewers, and authors are more diverse and that all authors receive the same fair treatment and opportunity to publish in our journals. We acknowledge that we do not have all the answers now, but we seek to hear from and listen to our community on how we can improve our journals to be more diverse and inclusive. As first steps, we commit to the taking the following actions: Gathering and making public our baseline statistics on diversity within our journals, encompassing our editors, advisors, reviewers, and authors; annually reporting on progress Training new and existing editors to recognize and interrupt bias in peer review Including diversity of journal contributors as an explicit measurement of Editor-in-Chief performance Appointing an ombudsperson to serve as a liaison between Editors and our Community Developing an actionable diversity plan for each ACS journal These are only initial plans and the start of a conversation: other ideas are beginning to germinate, and we commit to sharing them with you regularly. We invite you contribute your ideas on how we can do better via our Axial website. We are listening carefully. We encourage you to take immediate action in your own circles. In a recent editorial, JACS Associate Editor Melanie Sanford(6) offered practical steps to take now. Take a moment to find out more about these actions and how to bring them into your work and your life. We all have a responsibility to eradicate racism and discrimination in the science and engineering community; indeed, to make a real difference, we need to be antiracist. The tragic events we have seen in the Black community provide great urgency to this goal. The work will be difficult and will force us to confront hard realities about our beliefs and actions. We fully expect that you, and everyone in the community, will hold us accountable.

Maturation of mRNA in humans involves modifying the 5′ and 3′ ends, splicing introns, and installing epitranscriptomic modifications that are essential for mRNA biogenesis. With respect to epitranscriptomic modifications, they are usually installed in specific consensus motifs, although not all sequences are modified suggesting a secondary structural component to site selection. Using bioinformatic analysis of published data, we identify in human mature-mRNA that potential RNA G-quadruplex (rG4) sequences colocalize with the epitranscriptomic modifications N6-methyladenosine (m6A), pseudouridine (Ψ), and inosine (I). Using the only available pre-mRNA data sets from the literature, we demonstrate colocalization of potential rG4s and m6A was greatest overall and occurred in introns near 5′ and 3′ splice sites. The loop lengths and sequence context of the m6A-bearing potential rG4s exhibited short loops most commonly comprised of single A nucleotides. This observation is consistent with a literature report of intronic m6A found in SAG (S = C or G) consensus motifs that are also recognized by splicing factors. The localization of m6A and potential rG4s in pre-mRNA at intron splice junctions suggests that these features could function together in alternative splicing. A similar analysis for potential rG4s around sites of Ψ installation or A-to-I editing in mRNA also found a colocalization; however, the frequency was less than that observed with m6A. These bioinformatic analyses guide a discussion of future experiments to understand how noncanonical rG4 structures may collaborate with epitranscriptomic modifications in the human cellular context to impact cellular phenotype.

In DNA, i-motif (iM) folds occur under slightly acidic conditions when sequences rich in 2'-deoxycytidine (dC) nucleotides adopt consecutive dC self base pairs. The pH stability of an iM is defined by the midpoint in the pH transition (pHT ) between the folded and unfolded states. Two different experiments to determine pHT values via circular dichroism (CD) spectroscopy were performed on poly-dC iMs of length 15, 19, or 23 nucleotides. These experiments demonstrate two points: (1) pHT values were dependent on the titration experiment performed, and (2) pH-induced denaturing or annealing processes produced isothermal hysteresis in the pHT values. These results in tandem with model iMs with judicious mutations of dC to thymidine to favor particular folds found the hysteresis was maximal for the shorter poly-dC iMs and those with an even number of base pairs, while the hysteresis was minimal for longer poly-dC iMs and those with an odd number of base pairs. Experiments to follow the iM folding via thermal changes identified thermal hysteresis between the denaturing and annealing cycles. Similar trends were found to those observed in the CD experiments. The results demonstrate that the method of iM analysis can impact the pHT parameter measured, and hysteresis was observed in the pHT and Tm values.

Maturation of mRNA in humans involves modifying the 5' and 3' ends, splicing introns, and installing epitranscriptomic modifications that are essential for mRNA biogenesis. With respect to epitranscriptomic modifications, they are usually installed in specific consensus motifs, although not all sequences are modified suggesting a secondary structural component to site selection. Using bioinformatic analysis of published data, we identify in human mature-mRNA that potential RNA G-quadruplex (rG4) sequences colocalize with the epitranscriptomic modifications N6-methyladenosine (m6A), pseudouridine (Ψ), and inosine (I). Using the only available pre-mRNA data sets from the literature, we demonstrate colocalization of potential rG4s and m6A was greatest overall and occurred in introns near 5' and 3' splice sites. The loop lengths and sequence context of the m6A-bearing potential rG4s exhibited short loops most commonly comprised of single A nucleotides. This observation is consistent with a literature report of intronic m6A found in SAG (S = C or G) consensus motifs that are also recognized by splicing factors. The localization of m6A and potential rG4s in pre-mRNA at intron splice junctions suggests that these features could function together in alternative splicing. A similar analysis for potential rG4s around sites of Ψ installation or A-to-I editing in mRNA also found a colocalization; however, the frequency was less than that observed with m6A. These bioinformatic analyses guide a discussion of future experiments to understand how noncanonical rG4 structures may collaborate with epitranscriptomic modifications in the human cellular context to impact cellular phenotype.

Chlamydomonas reinhardtii is a green alga with a very GC-rich genome (67%) and a high density of potential G-quadruplex-forming sequences (PQSs). Using the Ensembl Plants DNA database, 19 PQSs were selected, and their ability to fold in vitro was examined using four experimental methods. Our results support in vitro folding of 18 of the 19 PQSs selected for study. The high physiological polyamine concentrations in C. reinhardtii create unique conditions for studying G4 folding. We investigated whether high polyamine concentrations affect the stability and structural fold of two polymorphic G4s selected from the cohort of PQSs. The two polymorphic G4s selected were found to be greatly stabilized when studied at the physiologically high polyamine concentrations. Lastly, the effects of physiologically relevant Mg2+ concentrations were tested on both of the polymorphic G4s, and one of the G4s shifted from a dynamic mixture of folds to favor a parallel fold in the presence of Mg2+. Our work supports the concept of folding of G4s under the unique conditions observed in C. reinhardtii, and these structures, being located in promoter regions of DNA repair and photosynthetic genes, might be relevant structures in the physiology of C. reinhardtii.

Biological nanopores provide a unique single-molecule sensing platform to detect target molecules based on their specific electrical signatures. The γ-hemolysin (γ-HL) protein produced by Staphylococcus aureus is able to assemble into an octamer nanopore with a ∼2.3 nm diameter β-barrel. Herein, we demonstrate the first application of γ-HL nanopore for DNA structural analysis. To optimize conditions for ion-channel recording, the properties of the γ-HL pore (e.g., conductance, voltage-dependent gating, and ion-selectivity) were characterized at different pH, temperature, and electrolyte concentrations. The optimal condition for DNA analysis using γ-HL corresponds to 3 M KCl, pH 5, and T = 20 °C. The γ-HL protein nanopore is able to translocate dsDNA at about ∼20 bp/ms, and the unique current-signature of captured dsDNA can directly distinguish guanine-to-inosine substitutions at the single-molecule level with ∼99% accuracy. The slow dsDNA threading and translocation processes indicate this wild-type γ-HL channel has potential to detect other base modifications in dsDNA.

We have studied the in vitro stability of 25 potential i-motif-forming DNA sequences found within the promoter regions of 18 different human DNA repair genes. Three widely available methods of characterization were used to rapidly assess i-motif folding and stability and comprise a simple screen for preliminary identification of physiologically relevant i-motif forming sequences. Four highly pH-stable candidate sequences were identified exhibiting pH transitions (pH at which 50% of the oligodeoxynucleotides in solution are folded) at or above pH 6.6, thermal melting temperatures above 37 °C and isothermal UV difference spectra characteristic of 2'-deoxycytidine imino-nitrogen protonation. These newly identified i-motif forming sequences could represent novel targets for understanding and modulating human DNA repair gene expression.

Our laboratory has recently proposed that the oxidation of guanine (G) to 8-oxo-7,8-dihydroguanine (OG) in G-rich promoter regions of DNA repair genes can serve as a regulatory mechanism of gene transcription. These regions also have the potential to fold into G-quadruplexes (G4). The human RAD17 promoter sequence has such a region in the template strand of the gene. In this work, the potential G-quadruplex sequence (PQS) of the RAD17 gene promoter was interrogated in different sequence contexts. With two extra nucleotides of the native sequence on either side of the G4, the structure was found to fold into a hybrid-like G4, similar to the hybrid-1 fold that the human telomere sequence can adopt. With only one nucleotide on either side of the PQS, the topology of the structure was observed to be mixed, and without extra nucleotides on the ends, the sequence adopted a parallel fold. Next, the sequence was studied with synthetic incorporation of the oxidative modification OG into specific sites and installed into the promoter of plasmids with a luciferase gene. These plasmids were transfected into a human cell line to observe the effect of the G4s on transcription. The RAD17 PQS was found to decrease luciferase expression with the presence of OG that is consistent with RAD17 expression under oxidative stress. This serves as an example of how oxidative modification could affect transcription in the context of a G4.

DNA oxidation is an inevitable and usually detrimental process, but the cell is capable of reversing this state because the cell possesses a highly developed set of DNA repair machineries, including the DNA glycosylase NEIL3 that is encoded by the NEIL3 gene. In this work, the G-rich promoter region of the human NEIL3 gene was shown to fold into a dynamic G-quadruplex (G4) structure under nearly physiological conditions using spectroscopic techniques (e.g., nuclear magnetic resonance, circular dichroism, fluorescence, and ultraviolet-visible) and DNA polymerase stop assays. The presence of 8-oxo-7,8-dihydroguanine (OG) modified the properties of the NEIL3 G4 and entailed the recruitment of the fifth domain to function as a "spare tire", in which an undamaged fifth G-track is swapped for the damaged section of the G4. The polymerase stop assay findings also revealed that owing to its dynamic polymorphism, the NEIL3 G4 is more readily bypassed by DNA polymerase I (Klenow fragment) than well-known oncogene G4s are. This study identifies the NEIL3 promoter possessing a G-rich element that can adopt a G4 fold, and when OG is incorporated, the sequence can lock into a more stable G4 fold via recruitment of the fifth track of Gs.