Nanopore sequencing for N1-methylpseudouridine in RNA reveals sequence-dependent discrimination of the modified nucleotide triphosphate during transcription.

Direct RNA sequencing with a commercial nanopore platform was used to sequence RNA containing uridine (U), pseudouridine (Ψ) or N1-methylpseudouridine (m1Ψ) in >100 different 5-nucleotide contexts. The base calling data for Ψ or m1Ψ were similar but different from U allowing their detection. Understanding the nanopore signatures for Ψ and m1Ψ enabled a running start T7 RNA polymerase assay to study the selection of UTP versus ΨTP or m1ΨTP competing mixtures in all possible adjacent sequence contexts. A significant sequence context dependency was observed for T7 RNA polymerase with insertion yields for ΨTP versus UTP spanning a range of 20-65%, and m1ΨTP versus UTP producing variable yields that differ by 15-70%. Experiments with SP6 RNA polymerase, as well as chemically-modified triphosphates and DNA templates provide insight to explain the observations. The SP6 polymerase introduced m1ΨTP when competed with UTP with a smaller window of yields (15-30%) across all sequence contexts studied. These results may aid in future efforts that employ RNA polymerases to make therapeutic mRNAs with sub-stoichiometric amounts of m1Ψ.

Fluorophore-mediated Photooxidation of the Guanine Heterocycle.

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 (1O2). The nucleoside reactions were then conducted in D2O solutions, known to increase the lifetime of 1O2, which resulted in a ~6-fold increase in the Sp yield, further supporting the classification of these dyes as Type II photosensitizers. Lastly, we inspected the pattern of G reactivity with the dyes upon photoirradiation in the context of a parallel-stranded G-quadruplex. The G nucleotides in the two exterior G-tetrads were found to be oxidation prone, providing the third line of evidence that the dyes are Type II photooxidants. The present work found that the common dyes fluorescein, rhodamine, BODIPY, or Cy3 can drive G oxidation but with a slow rate and low overall yield. This will likely not impact many experiments using dyes to study nucleic acids except for those that have long exposures with high-intensity lights, such as sequencing-by-synthesis experiments using fluorescence as the readout.

Second Harmonic Generation Interrogation of the Endonuclease APE1 Binding Interaction with G-Quadruplex DNA.

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 VEGF promoter G4 but whether this interaction was specific or not remained an open question. Second harmonic generation (SHG) was used in this work to measure the noncanonical DNA-protein binding interaction in a label-free assay with high sensitivity to demonstrate the interaction is ordered and specific. The binding of APE1 to the VEGF promoter G4 with AP sites modeled by a tetrahydrofuran analogue produced dissociation constants of ∼100 nM that differed from duplex and single-stranded DNA control studies. The SHG measurements confirmed APE1 binds the VEGF G4 folds in a specific manner resolving a remaining question regarding how this endonuclease with gene regulatory features engages G4 folds. The studies demonstrate the power of SHG to interrogate noncanonical DNA-protein interactions providing a foundational example for the use of this analytical method in future biochemical analyses.

Cysteine Oxidation to Sulfenic Acid in APE1 Aids G-Quadruplex Binding While Compromising DNA Repair.

Apurinic/apyrimidinic endonuclease-1 (APE1) is a base excision repair (BER) enzyme that is also engaged in transcriptional regulation. Previous work demonstrated that the enzymatic stalling of APE1 on a promoter G-quadruplex (G4) recruits transcription factors during oxidative stress for gene regulation. Also, during oxidative stress, cysteine (Cys) oxidation is a post-translational modification (PTM) that can change a protein's function. The current study provides a quantitative survey of cysteine oxidation to sulfenic acid in APE1 and how this PTM at specific cysteine residues affects the function of APE1 toward the NEIL3 gene promoter G4 bearing an abasic site. Of the seven cysteine residues in APE1, five (C65, C93, C208, C296, and C310) were prone to carbonate radical anion oxidation to yield sulfenic acids that were identified and quantified by mass spectrometry. Accordingly, five Cys-to-serine (Ser) mutants of APE1 were prepared and found to have attenuated levels of endonuclease activity, depending on the position, while KD values generally decreased for G4 binding, indicating greater affinity. These data support the concept that cysteine oxidation to sulfenic acid can result in modified APE1 that enhances G4 binding at the expense of endonuclease activity during oxidative stress. Cysteine oxidation to sulfenic acid residues should be considered as one of the factors that may trigger a switch from base excision repair activity to transcriptional modulation by APE1.

Pseudouridine and N1-Methylpseudouridine Display pH-Independent Reaction Rates with Bisulfite Yielding Ribose Adducts.

In RNA, pseudouridine (Ψ) and 5-methylcytidine (m5C) are located by their differential reactions with NaHSO3 at pH 5. The pyrimidines were allowed to react with NaHSO3, NaN3, NaCN, or NaSCN at pH 5 to find that NaHSO3 was unique in achieving quantitative yields. Pseudouridine reaction selectivity with NaHSO3 was found at pH 7 supported by the reaction rate constants. The Ψ derivative N1-methylpseudouridine found in mRNA vaccines reacts similarly with bisulfite to yield ribose adducts.

Insights into the 5-Carboxamido-5-Formamido-2-Iminohydantoin Structural Isomerization Equilibria.

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.

Identification of the Major Product of Guanine Oxidation in DNA by Ozone.

Ozonolysis of guanosine formed the 5-carboxamido-5-formamido-2-iminohydantoin (2Ih) nucleoside along with trace spiroiminodihydantoin (Sp). On the basis of literature precedent, we propose an unconventional ozone mechanism involving incorporation of only one oxygen atom of O3 to form 2Ih with evolution of singlet oxygen responsible for Sp formation. The increased yield of Sp in the buffered 1O2-stabilizing solvent D2O, formation of 2Ih in a short oligodeoxynucleotide, and 18O-isotope labeling provided evidence to support this mechanism. The elusiveness and challenges of working with 2Ih are described in a series of studies on the significant context effects on the half-life of the 2Ih glycosidic bond.

Riboflavin Stabilizes Abasic, Oxidized G-Quadruplex Structures.

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.

Chemistry of ROS-mediated oxidation to the guanine base in DNA and its biological consequences.

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.

Collateral Damage Occurs When Using Photosensitizer Probes to Detect or Modulate Nucleic Acid Modifications.

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 N6 -methyladenine (m6 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, m6 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 m6 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.

Nanopore Dwell Time Analysis Permits Sequencing and Conformational Assignment of Pseudouridine in SARS-CoV-2.

Direct RNA sequencing for the epitranscriptomic modification pseudouridine (Ψ), an isomer of uridine (U), was conducted with a protein nanopore sensor using a helicase brake to slowly feed the RNA into the sensor. Synthetic RNAs with 100% Ψ or U in 20 different known human sequence contexts identified differences during sequencing in the base-calling, ionic current, and dwell time in the nanopore sensor; however, the signals were found to have a dependency on the context that would result in biases when sequencing unknown samples. A solution to the challenge was the identification that the passage of Ψ through the helicase brake produced a long-range dwell time impact with less context bias that was used for modification identification. The data analysis approach was employed to analyze publicly available direct RNA sequencing data for SARS-CoV-2 RNA taken from cell culture to locate five conserved Ψ sites in the genome. Two sites were found to be substrates for pseudouridine synthase 1 and 7 in an in vitro assay, providing validation of the analysis. Utilization of the helicase as an additional sensor in direct RNA nanopore sequencing provides greater confidence in calling RNA modifications.

Oxidative stress-mediated epigenetic regulation by G-quadruplexes.

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.

Hysteresis in poly-2'-deoxycytidine i-motif folding is impacted by the method of analysis as well as loop and stem lengths.

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.

Binding of AP endonuclease-1 to G-quadruplex DNA depends on the N-terminal domain, Mg2+ and ionic strength.

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 that 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 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.

On the irrelevancy of hydroxyl radical to DNA damage from oxidative stress and implications for epigenetics.

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.