Reverse Transcription Past Products of Guanine Oxidation in RNA Leads to Insertion of A and C opposite 8‑Oxo-7,8-dihydroguanine and A and G opposite 5‑Guanidinohydantoin and Spiroiminodihydantoin Diastereomers

Reactive oxygen species, both endogenous and exogenous, can damage nucleobases of RNA and DNA. Among the nucleobases, guanine has the lowest redox potential, making it a major target of oxidation. Although RNA is more prone to oxidation than DNA is, oxidation of guanine in RNA has been studied to a significantly lesser extent. One of the reasons for this is that many tools that were previously developed to study oxidation of DNA cannot be used on RNA. In the study presented here, the lack of a method for seeking sites of modification in RNA where oxidation occurs is addressed. For this purpose, reverse transcription of RNA containing major products of guanine oxidation was used. Extension of a DNA primer annealed to an RNA template containing 8-oxo-7,8-dihydroguanine (OG), 5-guanidinohydantoin (Gh), or the <i>R</i> and <i>S</i> diastereomers of spiroiminodihydantoin (Sp) was studied under standing start conditions. SuperScript III reverse transcriptase is capable of bypassing these lesions in RNA inserting predominantly A opposite OG, predominantly G opposite Gh, and almost an equal mixture of A and G opposite the Sp diastereomers. These data should allow RNA sequencing of guanine oxidation products by following characteristic mutation signatures formed by the reverse transcriptase during primer elongation past G oxidation sites in the template RNA strand

Interactions of the Human Telomere Sequence with the Nanocavity of the α‑Hemolysin Ion Channel Reveal Structure-Dependent Electrical Signatures for Hybrid Folds

Human telomeric DNA consists of tandem repeats of the sequence 5′-TTAGGG-3′, including a 3′ terminal single-stranded overhang of 100–200 nucleotides that can fold into quadruplex structures in the presence of suitable metal ions. In the presence of an applied voltage, the α-hemolysin (α-HL) protein ion channel can produce unique current patterns that are found to be characteristic for various interactions between G-quadruplexes and the protein nanocavity. In this study, the human telomere in a complete sequence context, 5′-TAGGG­(TTAGGG)₃TT-3′, was evaluated with respect to its multiple folding topologies. Notably, the coexistence of two interchangeable conformations of the K⁺-induced folds, hybrid-1 and hybrid-2, were readily resolved at a single-molecule level along with triplex folding intermediates, whose characterization has been challenging in experiments that measure the bulk solution. These results enabled us to profile the thermal denaturation process of these structures to elucidate the relative distributions of hybrid-1, hybrid-2, and folding intermediates such as triplexes. For example, at 37 °C, pH 7.9, in 50 mM aqueous KCl, the ratio of hybrid-1:hybrid-2:triplex is approximately 11:5:1 in dilute solution. The results obtained lay the foundation for utilizing the α-HL ion channel as a simple tool for monitoring how small molecules and physical context shift the equilibrium between the many G-quadruplex folds of the human telomere sequence

Sequencing the Mouse Genome for the Oxidatively Modified Base 8‑Oxo-7,8-dihydroguanine by OG-Seq

Oxidative damage to the genome can yield the base 8-oxo-7,8-dihydroguanine (OG). In vitro studies suggested OG would preferentially form in 5′-GG-3′ sequence contexts after exposure to reactive oxygen species. Herein, OG locations in the genome were studied by development of “OG-Seq” to sequence OG sites via next-generation sequencing at ∼0.15-kb resolution. The results of this study found ∼10 000 regions of OG enrichment in WT mouse embryonic fibroblasts and ∼18 000 regions when the OG repair glycosylase Ogg1 was knocked out. Gene promoters and UTRs harbor more OG-enriched sites than expected if the sites were randomly distributed throughout the genome and correlate with reactive 5′-GG-3′ sequences, a result supporting decades of in vitro studies. Sequencing of OG paves the way to address chemical and biological questions surrounding this modified DNA base, such as its role in disease-specific mutations and its epigenetic potential in gene regulation

Sequencing of DNA Lesions Facilitated by Site-Specific Excision via Base Excision Repair DNA Glycosylases Yielding Ligatable Gaps

Modifications to nucleotides in the genome can lead to mutations or are involved in regulation of gene expression, and therefore, finding the site of modification is a worthy goal. Robust methods for sequencing modification sites on commercial sequencers have not been developed beyond the epigenetic marks on cytosine. Herein, a method to sequence DNA modification sites was developed that utilizes DNA glycosylases found in the base excision repair pathway to excise the modification. This approach yields a gap at the modification site that is sealed by T4-DNA ligase, yielding a product strand missing the modification. Upon sequencing, the modified nucleotide is reported as a deletion mutation, identifying its location. This approach was used to detect a uracil (U) or 8-oxo-7,8-dihydroguanine (OG) in codon 12 of the <i>KRAS</i> gene in synthetic oligo­deoxy­nucleotides. Additionally, an OG modification site was placed in the <i>VEGF</i> promoter in a plasmid and sequenced. This method requires only commercially available materials and can be put into practice on any sequencing platform, allowing this method to have broad potential for finding modifications in DNA

Human Telomere G‑Quadruplexes with Five Repeats Accommodate 8‑Oxo-7,8-dihydroguanine by Looping out the DNA Damage

Inflammation and oxidative stress generate free radicals that oxidize guanine (G) in DNA to 8-oxo-7,8-dihydroguanine (OG), and this reaction is prominent in the G-rich telomere sequence. In telomeres, OG is not efficiently removed by repair pathways allowing its concentration to build, surprisingly without any immediate negative consequences to stability. Herein, OG was synthesized in five repeats of the human telomere sequence (TTAGGG)_<i>n</i>, at the 5′-G of the 5′-most, middle, and 3′-most G tracks, representing hotspots for oxidation. These synthetic oligomers were folded in relevant amounts of K⁺/Na⁺ to adopt hybrid G-quadruplex folds. The structural impact of OG was assayed by circular dichroism, thermal melting, ¹H NMR, and single-molecule profiling by the α-hemolysin nanopore. On the basis of these results, OG was well accommodated in the five-repeat sequences by looping out the damaged G track to allow the other four tracks to adopt a hybrid G-quadruplex. These results run counter to previous studies with OG in four-repeat telomere sequences that found OG to be highly destabilizing and causing significant reorientation of the fold. When taking a wider view of the human telomere sequence and considering additional repeats, we found OG to cause minimal impact on the structure. The plasticity of this repeat sequence addresses how OG concentrations can increase in telomeres without immediate telomere instability or attrition

The Fifth Domain in the G‑Quadruplex-Forming Sequence of the Human <i>NEIL3</i> Promoter Locks DNA Folding in Response to Oxidative Damage

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 <i>NEIL3</i> gene. In this work, the G-rich promoter region of the human <i>NEIL3</i> 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 <i>NEIL3</i> 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 <i>NEIL3</i> G4 is more readily bypassed by DNA polymerase I (Klenow fragment) than well-known oncogene G4s are. This study identifies the <i>NEIL3</i> 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

Zika Virus Genomic RNA Possesses Conserved G‑Quadruplexes Characteristic of the Flaviviridae Family

Zika virus has emerged as a global concern because neither a vaccine nor antiviral compounds targeting it exist. A structure for the positive-sense RNA genome has not been established, leading us to look for potential G-quadruplex sequences (PQS) in the genome. The analysis identified >60 PQSs in the Zika genome. To minimize the PQS population, conserved sequences in the Flaviviridae family were found by sequence alignment, identifying seven PQSs in the prM, E, NS1, NS3, and NS5 genes. Next, alignment of 78 Zika strain genomes identified a unique PQS near the end of the 3′-UTR. Structural studies on the G-quadruplex sequences found four of the conserved Zika virus sequences to adopt stable, parallel-stranded folds that bind a G-quadruplex-specific compound, and one that was studied caused polymerase stalling when folded to a G-quadruplex. Targeting these PQSs with G-quadruplex binding molecules validated in previous clinical trials may represent a new approach for inhibiting viral replication

Nanopore Detection of 8‑Oxoguanine in the Human Telomere Repeat Sequence

The human telomere repeat sequence 5′-TTAGGG-3′ is a hot spot for oxidation at guanine, yielding 8-oxo-7,8-dihydroguanine (OG), a biomarker of oxidative stress. Telomere shortening resulting from oxidation will ultimately induce cellular senescence. In this study, α-hemolysin (α-HL) nanopore technology was applied to detect and quantify OG in the human telomeric DNA sequence. This repeat sequence adopts a basket G-quadruplex in the NaCl electrolyte used for analysis that enters the α-HL channel, slowly unfolds, and translocates. The basket fold containing OG disrupts the structure, leading to >10× increase in the unfolding kinetics without yielding a detectable current pattern. Therefore, detection of OG with α-HL required labeling of OG with aminomethyl-[18-crown-6] using a mild oxidant. The labeled OG yielded a pulse-like signal in the current <i>vs</i> time trace when the DNA strand was electrophoretically passed through α-HL in NaCl electrolyte. However, the rate of translocation was too slow using NaCl salts, leading us to further refine the method. A mixture of NH₄Cl and LiCl electrolytes induced the propeller fold that unravels quickly outside the α-HL channel. This electrolyte allowed observation of the labeled OG, while providing a faster recording of the currents. Lastly, OG distributions were probed with this method in a 120-mer stretch of the human telomere sequence exposed to the cellular oxidant ¹O₂. Single-molecule profiles determined the OG distributions to be random in this context. Application of the method in nanomedicine can potentially address many questions surrounding oxidative stress and telomere attrition observed in various disease phenotypes including prostate cancer and diabetes

Unfolding Kinetics of the Human Telomere i‑Motif Under a 10 pN Force Imposed by the α‑Hemolysin Nanopore Identify Transient Folded-State Lifetimes at Physiological pH

Cytosine (C)-rich DNA can adopt i-motif folds under acidic conditions, with the human telomere i-motif providing a well-studied example. The dimensions of this i-motif are appropriate for capture in the nanocavity of the α-hemolysin (α-HL) protein pore under an electrophoretic force. Interrogation of the current vs time (<i>i</i>–<i>t</i>) traces when the i-motif interacts with α-HL identified characteristic signals that were pH dependent. These features were evaluated from pH 5.0 to 7.2, a region surrounding the transition pH of the i-motif (6.1). When the i-motif without polynucleotide tails was studied at pH 5.0, the folded structure entered the nanocavity of α-HL from either the top or bottom face to yield characteristic current patterns. Addition of a 5′ 25-mer poly-2′-deoxyadensosine tail allowed capture of the i-motif from the unfolded terminus, and this was used to analyze the pH dependency of unfolding. At pH values below the transition point, only folded strands were observed, and when the pH was increased above the transition pH, the number of folded events decreased, while the unfolded events increased. At pH 6.8 and 7.2 4% and 2% of the strands were still folded, respectively. The lifetimes for the folded states at pH 6.8 and 7.2 were 21 and 9 ms, respectively, at 160 mV electrophoretic force. These lifetimes are sufficiently long to affect enzymes operating on DNA. Furthermore, these transient lifetimes are readily obtained using the α-HL nanopore, a feature that is not easily achievable by other methods

Unzipping Kinetics of Duplex DNA Containing Oxidized Lesions in an α-Hemolysin Nanopore

The unzipping kinetics for lesion-containing DNA duplexes was studied in an α-hemolysin (α-HL) nanopore. The lesion of focus was the guanine two-electron oxidation product, 8-oxo-7,8-dihydroguanine (OG), and its further oxidation products, the hydantoins guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp). The voltage-driven unzipping of individual duplex DNA molecules with symmetrical overhangs was carried out by pulling one strand of the duplex through the α-HL channel using an electrical field. Entry from the 3′ or 5′ end produced distinct current blockages, allowing directional effects on unzipping kinetics to be investigated. We find that the strand dissociation of complementary duplexes or duplexes containing the slightly destabilizing lesion OG follows a first-order kinetic model, while opening of duplexes that contain the highly destabilizing lesions Gh or Sp is described by two sequential first-order reactions, in which the intermediate state is proposed to correspond to the duplex unzipped to the lesion site within the channel. The rate constants for strand separation of the duplexes containing single lesions were obtained from kinetic model fits to histograms of unzipping duration. For all duplexes, the rate constants for strand separation displayed a significant dependence on the direction of entry into the nanopore. For duplexes containing Gh, truncated duplexes were used to assign the measured rate constants for the first and second unzipping steps of symmetrically designed duplexes