An α-ketoglutarate conformational switch controls iron accessibility, activation, and substrate selection of the human FTO protein.

The fat mass and obesity-associated fatso (FTO) protein is a member of the Alkb family of dioxygenases and catalyzes oxidative demethylation of N6-methyladenosine (m6A), N1-methyladenosine (m1A), 3-methylthymine (m3T), and 3-methyluracil (m3U) in single-stranded nucleic acids. It is well established that the catalytic activity of FTO proceeds via two coupled reactions. The first reaction involves decarboxylation of alpha-ketoglutarate (αKG) and formation of an oxyferryl species. In the second reaction, the oxyferryl intermediate oxidizes the methylated nucleic acid to reestablish Fe(II) and the canonical base. However, it remains unclear how binding of the nucleic acid activates the αKG decarboxylation reaction and why FTO demethylates different methyl modifications at different rates. Here, we investigate the interaction of FTO with 5-mer DNA oligos incorporating the m6A, m1A, or m3T modifications using solution NMR, molecular dynamics (MD) simulations, and enzymatic assays. We show that binding of the nucleic acid to FTO activates a two-state conformational equilibrium in the αKG cosubstrate that modulates the O2 accessibility of the Fe(II) catalyst. Notably, the substrates that provide better stabilization to the αKG conformation in which Fe(II) is exposed to O2 are demethylated more efficiently by FTO. These results indicate that i) binding of the methylated nucleic acid is required to expose the catalytic metal to O2 and activate the αKG decarboxylation reaction, and ii) the measured turnover of the demethylation reaction (which is an ensemble average over the entire sample) depends on the ability of the methylated base to favor the Fe(II) state accessible to O2.

Decarboxylation Mechanism of iso-Orotate Decarboxylase Revisited.

iso-Orotate decarboxylase (IDCase), which is involved in the thymidine salvage pathway, has attracted considerable interest owing to its chemical similarity to a hypothetical DNA decarboxylase in mammals. Although valuable insights into the active DNA demethylation of 5-methyl-cytosine can be obtained from the decarboxylation mechanism of 5-carboxyl-uracil (5caU) catalyzed by IDCase, this mechanism remains under debate. In this study, the catalytic mechanism of 5caU decarboxylation by IDCase was studied using hybrid quantum mechanics/molecular mechanics (QM/MM) methodologies and density functional theory (DFT) calculations with a truncated model. The calculations supported a mechanism involving three sequential stages: activation of the 5caU substrate via proton transfer from an arginine (R262') to the carboxyl group of 5caU, formation of a tetrahedral intermediate, and decarboxylation of the tetrahedral intermediate to generate uracil as the product. The reaction pathways and structures obtained using the QM/MM and DFT methods coincided with each other. These simulations provided detailed insights into the unique mechanism of IDCase, clarifying various unresolved issues, such as the critical role of R262'. In addition, aspartate D323 was found to act as a general base in the tetrahedral intermediate formation step and a general acid in the later C-C bond cleavage step.

Properties of chromatographic columns prepared on the basis of poly(1-trimethylsilyl-1-propyne) modified with organic bases.

This article describes the effect of modification with organic bases such as uracil (U) and polyethyleneimine (PEI) on the adsorption and chromatographic properties of poly(1-trimethylsilyl-1-propyne) (PTMSP) used as a stationary phase (SP) in packed and capillary columns. It was shown that the sorbents prepared on the basis of diatomite Chromosorb P NAW support and successively modified with 9 wt.% PTMSP and 1 wt.% U (or PEI) (PC-U and PC-PEI samples, respectively), have a mesoporous structure. The IR spectrum shows the presence of carbonyl groups in the sorbent modified with uracil. The impregnation of the Chromosorb P NAW + (9/1) wt.% PTMSP sorbent with a polyethyleneimine solution leads to the appearance in the spectrum of bands characterizing NH stretching and bending vibrations, as well as a band at 1310 cm-1 which can be attributed to CN bond vibrations. The chromatographic properties of the studied sorbents differ significantly from the properties of the initial PTMSP. Packed columns PC-U and PC-PEI, as well as capillary columns with a polyethyleneimine-modified PTMSP layer, allow one to selectively separate mixtures of polar and non-polar compounds and structural isomers of hydrocarbons. Methanol on these columns is eluted in the form of a symmetrical peak separately from propane, propylene and other associated hydrocarbon impurities in commercial (technical, target) n-butane.

Reaction sites of pyrimidine bases and nucleosides during chlorination: A computational study.

As important components of soluble microbial products in water, nucleobases have attracted much attention due to the high toxicity of their direct aromatic halogenated disinfection by-products (AH-DBPs) during chlorination. However, multiple halogenation sites of AH-DBPs pose challenges to identify them. In this study, reaction sites of pyrimidine bases and nucleosides during chlorination were investigated by quantum chemical computational method. The results indicate that the anion salt forms play key roles in chlorination of uracil, thymine, and their nucleosides, while neutral forms make predominant contributions to cytosine and cytidine. In view of both kinetics and thermodynamics, C5 is the most reactive site for uracil and thymine, N3/C5 and N3 for respective uridine and thymidine, N1/C5/N4 and N4 for respective cytosine and cytidine, whose estimated apparent rate constants kobs-est of ∼103, 103/102, 106/102/104, and 103 M-1 s-1, respectively, in consistent with the known experimental results. C6 in all pyrimidine compounds is hardly attacked by Cl+ in HOCl ascribed to its positive charge, but readily attacked by OH‾ in hydrolysis and the N1=C6 bond was found to possess the highest reactivity in hydrolysis among all double bonds. In addition, the structure-kinetic reactivity relationship study reveals a relatively strong correlation between lgkobs-est and APT charge in all pyrimidine compounds rather than FED2 (HOMO). The results are helpful to further understand the reactivity of various reaction sites in aromatic compounds during chlorination.

Mouse mucosal-associated invariant T cell receptor recognition of MR1 presenting the vitamin B metabolite, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil.

Mucosal-associated invariant T (MAIT) cells can elicit immune responses against riboflavin-based antigens presented by the evolutionary conserved MHC class I related protein, MR1. While we have an understanding of the structural basis of human MAIT cell receptor (TCR) recognition of human MR1 presenting a variety of ligands, how the semi-invariant mouse MAIT TCR binds mouse MR1-ligand remains unknown. Here, we determine the crystal structures of 2 mouse TRAV1-TRBV13-2+ MAIT TCR-MR1-5-OP-RU ternary complexes, whose TCRs differ only in the composition of their CDR3ß loops. These mouse MAIT TCRs mediate high affinity interactions with mouse MR1-5-OP-RU and cross-recognize human MR1-5-OP-RU. Similarly, a human MAIT TCR could bind mouse MR1-5-OP-RU with high affinity. This cross-species recognition indicates the evolutionary conserved nature of this MAIT TCR-MR1 axis. Comparing crystal structures of the mouse versus human MAIT TCR-MR1-5-OP-RU complexes provides structural insight into the conserved nature of this MAIT TCR-MR1 interaction and conserved specificity for the microbial antigens, whereby key germline-encoded interactions required for MAIT activation are maintained. This is an important consideration for the development of MAIT cell-based therapeutics that will rely on preclinical mouse models of disease.

Gene identification and enzymatic characterization of the initial enzyme in pyrimidine oxidative metabolism, uracil-thymine dehydrogenase.

Uracil-thymine dehydrogenase (UTDH), which catalyzes the irreversible oxidation of uracil to barbituric acid in oxidative pyrimidine metabolism, was purified from Rhodococcus erythropolis JCM 3132. The finding of unusual stabilizing conditions (pH 11, in the presence of NADP+ or NADPH) enabled the enzyme purification. The purified enzyme was a heteromer consisting of three different subunits. The enzyme catalyzed oxidation of uracil to barbituric acid with artificial electron acceptors such as methylene blue, phenazine methosulfate, benzoquinone, and α-naphthoquinone; however, NAD+, NADP+, flavin adenine dinucleotide, and flavin mononucleotide did not serve as electron acceptors. The enzyme acted not only on uracil and thymine but also on 5-halogen-substituted uracil and hydroxypyrimidine (pyrimidone), while dihydropyrimidine, which is an intermediate in reductive pyrimidine metabolism, and purine did not serve as substrates. The activity of UTDH was enhanced by cerium ions, and this activation was observed with all combinations of substrates and electron acceptors.

Test strip coupled Cas12a-assisted signal amplification strategy for sensitive detection of uracil-DNA glycosylase.

Uracil-DNA glycosylase (UDG) is a base excision repair (BER) enzyme, which catalyzes the hydrolysis of uracil bases in DNA chains that contain uracil and N-glycosidic bonds of the sugar phosphate backbone. The expression of UDG enzyme is associated with a variety of genetic diseases including cancers. Hence, the identification of UDG activity in cellular processes holds immense importance for clinical investigation and diagnosis. In this study, we employed Cas12a protein and enzyme-assisted cycle amplification technology with a test strip to establish a precise platform for the detection of UDG enzyme. The designed platform enabled amplifying and releasing the target probe by reacting with the UDG enzyme. The amplified target probe can subsequently fuse with crRNA and Cas12a protein, stimulating the activation of the Cas12a protein to cleave the signal probe, ultimately generating a fluorescent signal. This technique showed the ability for evaluating UDG enzyme activity in different cell lysates. In addition, we have designed a detection probe to convert the fluorescence signal into test strip bands that can then be observed with the naked eye. Hence, our tool presented potential in both biomedical research and clinical diagnosis related to DNA repair enzymes.

Electron interaction with DNA constituents in aqueous phase.

Electron driven chemistry of biomolecules in aqueous phase presents the realistic picture to study molecular processes. In this study we have investigated the interactions of electrons with the DNA constituents in their aqueous phase in order to obtain the quantities useful for DNA damage assessment. We have computed the inelastic mean free path (IMFP), mass stopping power (MSP) and absorbed dose (D) for the DNA constituents (Adenine, Cytosine, Guanine, Thymine and Uracil) in the aqueous medium from ionisation threshold to 5000 eV. We have modified complex optical potential formalism to include band gap of the systems to calculate inelastic cross sections which are used to estimate these entities. This is the maiden attempt to report these important quantities for the aqueous DNA constituents. We have compared our results with available data in gas and other phase and have observed explicable accord for IMFP and MSP. Since these are the first results of absorbed dose (D) for these compounds, we have explored present results vis-a-vis dose absorption in water.

Chimeric GFP-uracil based molecular rotor fluorophores.

Uracil has been modified at the 5-position to derive a small library of nucleobase-chromophores which were inspired by green fluorescent protein (GFP). The key steps in the syntheses were Erlenmeyer azlactone synthesis followed by amination by use of hexamethyl disilazane (HMDS) to produce the imidazolinone derivatives. The uracil analogues displayed emission in the green region of visible spectrum and exhibited microenvironmental sensitivity exemplified by polarity-based solvatochromism and viscosity-dependent emission enhancement. Solid-state quantum yields of approximately 0.2 and solvent dependent emission wavelengths beyond 500 nm were observed. Select analogues were incorporated into peptide nucleic acid (PNA) strands which upon duplex formation with DNA showed good response ranging from a turn-off of fluorescence in presence of an opposing mismatched residue to a greater than 3-fold turn-on of fluorescence upon binding to fully complementary DNA strand.

Register-Shifted Structures in Base-Flipped Uracil-Damaged DNA.

We report the occurrence of register-shifted structures in simulations of uracil-containing dsDNA. These occur when the 3' base vicinal to uracil is thymine in U:A base-paired DNA. Upon base flipping of uracil, this 3' thymine hydrogen bonds with the adenine across the uracil instead of its complementary base. The register-shifted structure is persistent and sterically blocks re-entry of uracil into the helix stack. Register shifting might be important for DNA repair since the longer exposure of the lesion in register-shifted structures could facilitate enzymatic recognition and repair.

Escherichia coli DNA repair helicase Lhr is also a uracil-DNA glycosylase.

DNA glycosylases protect genetic fidelity during DNA replication by removing potentially mutagenic chemically damaged DNA bases. Bacterial Lhr proteins are well-characterized DNA repair helicases that are fused to additional 600-700 amino acids of unknown function, but with structural homology to SecB chaperones and AlkZ DNA glycosylases. Here, we identify that Escherichia coli Lhr is a uracil-DNA glycosylase (UDG) that depends on an active site aspartic acid residue. We show that the Lhr DNA helicase activity is functionally independent of the UDG activity, but that the helicase domains are required for fully active UDG activity. Consistent with UDG activity, deletion of lhr from the E. coli chromosome sensitized cells to oxidative stress that triggers cytosine deamination to uracil. The ability of Lhr to translocate single-stranded DNA and remove uracil bases suggests a surveillance role to seek and remove potentially mutagenic base changes during replication stress.

Distant sequence regions of JBP1 contribute to J-DNA binding.

Base-J (ß-D-glucopyranosyloxymethyluracil) is a modified DNA nucleotide that replaces 1% of thymine in kinetoplastid flagellates. The biosynthesis and maintenance of base-J depends on the base-J-binding protein 1 (JBP1) that has a thymidine hydroxylase domain and a J-DNA-binding domain (JDBD). How the thymidine hydroxylase domain synergizes with the JDBD to hydroxylate thymine in specific genomic sites, maintaining base-J during semi-conservative DNA replication, remains unclear. Here, we present a crystal structure of the JDBD including a previously disordered DNA-contacting loop and use it as starting point for molecular dynamics simulations and computational docking studies to propose recognition models for JDBD binding to J-DNA. These models guided mutagenesis experiments, providing additional data for docking, which reveals a binding mode for JDBD onto J-DNA. This model, together with the crystallographic structure of the TET2 JBP1-homologue in complex with DNA and the AlphaFold model of full-length JBP1, allowed us to hypothesize that the flexible JBP1 N-terminus contributes to DNA-binding, which we confirmed experimentally. Α high-resolution JBP1:J-DNA complex, which must involve conformational changes, would however need to be determined experimentally to further understand this unique underlying molecular mechanism that ensures replication of epigenetic information.

Incorporation of a bromine atom into DNA-related molecules changes their electronic properties.

To understand the mechanism underlying the high radio-sensitisation of living cells possessing brominated genomic DNA, X-ray photoelectron spectroscopy (XPS) using synchrotron X-rays with energies of 2000 or 2500 eV was used to study brominated and nonbrominated nucleobases, nucleosides and nucleotides. The bromine atom significantly reduced the energy gap between the valence and conduction states, although the core level states were not greatly affected. This finding was supported by quantum chemical calculation for the nucleobases and nucleosides. Our findings strongly indicate that the energy gaps between the valence and conduction levels of the molecules are significantly reduced by bromination. Furthermore, the brominated molecules are more likely to produce inelastic scattering low energy electrons upon exposure to 2000 or 3000 eV X-rays. This modification of electronic properties around the brominated group may both facilitate electron transfer to the brominated site in DNA and increase the probability of reaction with low energy electrons. These processes can induce DNA damage, presumably resulting in debromination of the uracil moiety and a subsequent cytotoxic effect.

Enzymatic Cleavage-Mediated Extension Stalling Enables Accurate Recognition and Quantification of Locus-Specific Uracil Modification in DNA.

Chemical modifications in DNA have profound influences on the structures and functions of DNA. Uracil, a naturally occurring DNA modification, can originate from the deamination of cytosine or arise from misincorporation of dUTP into DNA during DNA replication. Uracil in DNA will imperil genomic stability due to their potential in producing detrimental mutations. An in-depth understanding of the functions of uracil modification requires the accurate determination of its site as well as content in genomes. Herein, we characterized that a new member of the uracil-DNA glycosylase (UDG) family enzyme (UdgX-H109S) could selectively cleave both uracil-containing single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). Based on this unique property of UdgX-H109S, we developed an enzymatic cleavage-mediated extension stalling (ECES) method for the locus-specific detection and quantification of uracil in genomic DNA. In the ECES method, UdgX-H109S specifically recognizes and cleaves the N-glycosidic bond of uracil from dsDNA and generates an apurinic/apyrimidinic (AP) site, which could be broken by APE1 to form a one-nucleotide gap. The specific cleavage by UdgX-H109S is then evaluated and quantified by qPCR. With the developed ECES approach, we demonstrated that the level of uracil at position Chr4:50566961 in genomic DNA of breast cancer tissues was significantly decreased. Collectively, the ECES method has been proved to be accurate and reproducible in the locus-specific quantification of uracil in genomic DNA from biological and clinical samples.

Prodrug Strategies for the Development of ß-l-5-((E)-2-Bromovinyl)-1-((2S,4S)-2-(hydroxymethyl)-1,3-(dioxolane-4-yl))uracil (l-BHDU) against Varicella Zoster Virus (VZV).

Varicella zoster virus (VZV) establishes lifelong infection after primary disease and can reactivate. Several drugs are approved to treat VZV diseases, but new antivirals with greater potency are needed. Previously, we identified ß-l-5-((E)-2-bromovinyl)-1-((2S,4S)-2-(hydroxymethyl)-1,3-(dioxolane-4-yl))uracil (l-BHDU, 1), which had significant anti-VZV activity. In this communication, we report the synthesis and evaluation of numerous l-BHDU prodrugs: amino acid esters (14-26), phosphoramidates (33-34), long-chain lipids (ODE-l-BHDU-MP, 38, and HDP-l-BHDU-MP, 39), and phosphate ester prodrugs (POM-l-BHDU-MP, 41, and POC-l-BHDU-MP, 47). The amino acid ester l-BHDU prodrugs (l-phenylalanine, 16, and l-valine, 17) had a potent antiviral activity with EC50 values of 0.028 and 0.030 µM, respectively. The phosphate ester prodrugs POM-l-BHDU-MP and POC-l-BHDU-MP had a significant anti-VZV activity with EC50 values of 0.035 and 0.034 µM, respectively, and no cellular toxicity (CC50 > 100 µM) was detected. Out of these prodrugs, ODE-l-BHDU-MP (38) and POM-l-BHDU-MP (41) were selected for further evaluation in future studies.

Ferrate(VI) mediated degradation of the potent cyanotoxin, cylindrospermopsin: Kinetics, products, and toxicity.

The presence of cylindrospermopsin (CYN), a potent cyanotoxin, in drinking water sources poses a tremendous risk to humans and the environment. Detailed kinetic studies herein demonstrate ferrate(VI) (FeVIO42-, Fe(VI)) mediated oxidation of CYN and the model compound 6-hydroxymethyl uracil (6-HOMU) lead to their effective degradation under neutral and alkaline solution pH. A transformation product analysis indicated oxidation of the uracil ring, which has functionality critical to the toxicity of CYN. The oxidative cleavage of the C5=C6 double bond resulted in fragmentation of the uracil ring. Amide hydrolysis is a contributing pathway leading to the fragmentation of the uracil ring. Under extended treatment, hydrolysis, and extensive oxidation lead to complete destruction of the uracil ring skeleton, resulting in the generation of a variety of products including nontoxic cylindrospermopsic acid. The ELISA biological activity of the CYN product mixtures produced during Fe(VI) treatment parallels the concentration of CYN. These results suggest the products do not possess ELISA biological activity at the concentrations produced during treatment. The Fe(VI) mediated degradation was also effective in the presence of humic acid and unaffected by the presence of common inorganic ions under our experimental conditions. The Fe(VI) remediation of CYN and uracil based toxins appears a promising drinking water treatment process.

Photoreactive Mercury-Containing Metallosupramolecular Nanoparticles with Tailorable Properties That Promote Enhanced Cellular Uptake for Effective Cancer Chemotherapy.

A new potential route to enhance the efficiency of supramolecular polymers for cancer chemotherapy was successfully demonstrated by employing a photosensitive metallosupramolecular polymer (Hg-BU-PPG) containing an oligomeric poly(propylene glycol) backbone and highly sensitive pH-responsive uracil-mercury-uracil (U-Hg-U) bridges. This route holds great promise as a multifunctional bioactive nano-object for development of more efficient and safer cancer chemotherapy. Owing to the formation of uracil photodimers induced by ultraviolet irradiation, Hg-BU-PPG can form a photo-cross-linked structure and spontaneously forms spherical nanoparticles in aqueous solution. The irradiated nanoparticles possess many unique characteristics, such as unique fluorescence behavior, highly sensitive pH-responsiveness, and intriguing phase transition behavior in aqueous solution as well as high structural stability and antihemolytic activity in biological media. More importantly, a series of cellular studies clearly confirmed that the U-Hg-U photo-cross-links in the irradiated nanoparticles substantially enhance their selective cellular uptake by cancer cells via macropinocytosis and the mercury-loaded nanoparticles subsequently induce higher levels of cytotoxicity in cancer cells (compared to non-irradiated nanoparticles), without harming normal cells. These results are mainly attributed to cancer cell microenvironment-triggered release of mercury ions from disassembled nanoparticles, which rapidly induce massive levels of apoptosis in cancer cells. Overall, the pH-sensitive U-Hg-U photo-cross-links within this newly discovered supramolecular system are an indispensable factor that offers a potential path to remarkably enhance the selective therapeutic effects of functional nanoparticles toward cancer cells.

Low-Energy Shape Resonances of a Nucleobase in Water.

When high-energy radiation passes through aqueous material, low-energy electrons are produced which cause DNA damage. Electronic states of anionic nucleobases have been suggested as an entrance channel to capture the electron. However, identifying these electronic resonances have been restricted to gas-phase electron-nucleobase studies and offer limited insight into the resonances available within the aqueous environment of DNA. Here, resonance and detachment energies of the micro-hydrated uracil pyrimidine nucleobase anion are determined by two-dimensional photoelectron spectroscopy and are shown to extrapolate linearly with cluster size. This extrapolation allows the corresponding resonance and detachment energies to be determined for uracil in aqueous solution as well as the reorganization energy associated with electron capture. Two shape resonances are clearly identified that can capture low-energy electrons and subsequently form the radical anion by solvent stabilization and internal conversion to the ground electronic state. The resonances and their dynamics probed here are the nucleobase-centered doorway states for low-energy electron capture and damage in DNA.

Intramolecular Interactions in Derivatives of Uracil Tautomers.

The influence of solvents on intramolecular interactions in 5- or 6-substituted nitro and amino derivatives of six tautomeric forms of uracil was investigated. For this purpose, the density functional theory (B97-D3/aug-cc-pVDZ) calculations were performed in ten environments (1 > ε > 109) using the polarizable continuum model (PCM) of solvation. The substituents were characterized by electronic (charge of the substituent active region, cSAR) and geometric parameters. Intramolecular interactions between non-covalently bonded atoms were investigated using the theory of atoms in molecules (AIM) and the non-covalent interaction index (NCI) method, which allowed discussion of possible interactions between the substituents and N/NH endocyclic as well as =O/−OH exocyclic groups. The nitro group was more electron-withdrawing in the 5 than in the 6 position, while the opposite effect was observed in the case of electron donation of the amino group. These properties of both groups were enhanced in polar solvents; the enhancement depended on the ortho interactions. Substitution or solvation did not change tautomeric preferences of uracil significantly. However, the formation of a strong NO∙∙∙HO intramolecular hydrogen bond in the 5-NO2 derivative stabilized the dienol tautomer from +17.9 (unsubstituted) to +5.4 kcal/mol (substituted, energy relative to the most stable diketo tautomer).

Building the uracil skeleton in primitive ponds at the origins of life: carbamoylation of aspartic acid.

A large set of nucleobases and amino acids is found in meteorites, implying that several chemical reservoirs are present in the solar system. The "geochemical continuity" hypothesis explores how protometabolic paths developed from so-called "bricks" in an enzyme-free prebiotic world and how they affected the origins of life. In the living cell, the second step of synthesizing uridine and cytidine RNA monomers is a carbamoyl transfer from a carbamoyl donor to aspartic acid. Here we compare two enzyme-free scenarios: aqueous and mineral surface scenarios in a thermal range up to 250 °C. Both processes could have happened in ponds under open atmosphere on the primeval Earth. Carbamoylation of aspartic acid with cyanate in aqueous solutions at 25 °C gives high N-carbamoyl aspartic acid yields within 16 h. It is important to stress that, while various molecules could be efficient carbamoylating agents according to thermodynamics, kinetics plays a determining role in selecting prebiotically possible pathways.