Methylated Nucleic Acid Research Articles

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.

Dual-modality loop-mediated isothermal amplification for pretreatment-free detection of Septin9 methylated DNA in colorectal cancer.

Currently, the determination of DNA methylation is still a challenge due to the limited efficiency of enrichment, bisulfite modification, and detection. In this study, a dual-modality loop-mediated isothermal amplification integrated with magnetic bead isolation is proposed for the determination of methylated Septin9 gene in colorectal cancer. Magnetic beads modified with anti-methyl cytosine antibody were prepared for fast enrichment of methylated DNA through specific immunoaffinity (30 min). One-pot real-time fluorescence and colorimetric loop-mediated isothermal amplification were simultaneously developed for detecting methylated Septin9 gene (60 min). The real-time fluorescence generating by SYTO-9 dye (excitation: 470 nm and emission: 525 nm) and pH indicator (neutral red) was used for quantitative and visualized detection of methylated DNA. This method was demonstrated to detect methylated DNA from HCT 116 cells ranging from 2 to 0.02 ng/μL with a limit of detection of 0.02 ± 0.002 ng/μL (RSD: 9.75%). This method also could discriminate methylated Septin9 in 0.1% HCT 116 cells (RSD: 6.60%), suggesting its high specificity and sensitivity. The feasibility of this assay was further evaluated by clinical plasma samples from 20 colorectal cancer patients and 20 healthy controls, which shows the potential application in simple, low cost, quantitative, and visualized detection of methylated nucleic acids.Graphical abstract A dual-modality loop-mediated isothermal amplification (LAMP) integrated with immuno-magnetic beads (IMB) enrichment was proposed for the determination of methylated Septin9 gene in colorectal cancer (CRC).Supplementary InformationThe online version contains supplementary material available at 10.1007/s00604-021-04979-8.

Methylated Polytope of Hereditary Information

The geometric features of nucleic acids during methylation of their nitrogenous bases, which is typical for viral nucleic acids in infectious diseases, were studied. It was found that methylation of viral nucleic acids leads to the transition of their nitrogenous bases into the space of higher dimension and sharply increases (more than 2000 times) the intensity of information processes in the field of interaction of nucleic acids (in the polytopic of hereditary information). The activity of methylated viral nucleic acids can be countered by activation (i.e., a fairly high degree of methylation, nucleic acids of the organism).

Degree of N-Methylation of Nucleosides and Metabolites Controls Binding Affinity to Pristine Silica Surfaces.

Biological molecules interact with silica (SiO2) surfaces with binding affinities that greatly vary depending on their physical-chemical properties. However, the quantitative characterization of biological compounds adsorbed on silica surfaces, especially of compounds involved in fast, reversible interactions, has been challenging, and the driving forces are not well understood. Here, we show how carbon-13 NMR spin relaxation provides quantitative atomic-detail information about the transient molecular binding to pristine silica surfaces, represented by colloidally dispersed silica nanoparticles (SNPs). Based on the quantitative analysis of almost two dozen biological molecules, we find that the addition of N-methyl motifs systematically increases molecular binding affinities to silica in a nearly quantitatively predictable manner. Among the studied compounds are methylated nucleosides, which are common in epigenetic signaling in nucleic acids. The quantitative understanding of N-methylation may open up new ways to detect and separate methylated nucleic acids or even regulate their cellular functions.

The GDF11-FTO-PPARγ axis controls the shift of osteoporotic MSC fate to adipocyte and inhibits bone formation during osteoporosis

During osteoporosis, the shift of bone mesenchymal stem cell (BMSC) lineage commitment to adipocyte leads to the imbalance between bone mass and fat, which increases the risk of fracture. The mechanism underlying this process is not fully understood. Fat mass and obesity-associated protein (FTO) is an RNA demethylase that demethylates various methylated nucleic acids and participates in various physiological and pathological processes. Here we identified FTO as a regulator for BMSC fate determination during osteoporosis. FTO was up-regulated in bone marrow during aging or osteoporosis in human and mice in a GDF11(growth differentiation factor 11)-C/EBPα-dependent mechanism. The expression of FTO was also up-regulated during adipocyte differentiation of BMSCs whereas its expression was down-regulated during osteoblast differentiation. Gain-of-function and loss-of-function experiments showed that FTO favored the BMSCs to differentiate to adipocytes rather than osteoblasts. Further mechanism study demonstrated that FTO bound and demethylated the mRNA of the Peroxisome proliferator-activated receptor gamma (Pparg), leading to the increase in the expression of Pparg mRNA. Reversely, Pparg knockdown blocked the function of GDF11-FTO during osteoblast differentiation of BMSCs. Furthermore, conditionally genetic knockout of Fto in osteoblasts inhibited the development of osteopenia in mice. Collectively, our findings demonstrated that GDF11-FTO-Pparg axis promoted the shift of osteoporotic BMSC fate to adipocyte and inhibited bone formation during osteoporosis.

Comparative Structural Dynamics of tRNA(Phe) with Respect to Hinge Region Methylated Guanosine: A Computational Approach.

Transfer RNAs (tRNAs) contain various uniquely modified nucleosides thought to be useful for maintaining the structural stability of tRNAs. However, their significance for upholding the tRNA structure has not been investigated in detail at the atomic level. In this study, molecular dynamic simulations have been performed to assess the effects of methylated nucleic acid bases, N (2)-methylguanosine (m(2)G) and N (2)-N (2)-dimethylguanosine (m 2 (2) G) at position 26, i.e., the hinge region of E. coli tRNA(Phe) on its structure and dynamics. The results revealed that tRNA(Phe) having unmodified guanosine in the hinge region (G26) shows structural rearrangement in the core of the molecule, resulting in lack of base stacking interactions, U-turn feature of the anticodon loop, and TΨC loop. We show that in the presence of the unmodified guanosine, the overall fold of tRNA(Phe) is essentially not the same as that of m(2)G26 and m 2 (2) G26 containing tRNA(Phe). This structural rearrangement arises due to intrinsic factors associated with the weak hydrogen-bonding patterns observed in the base triples of the tRNA(Phe) molecule. The m(2)G26 and m 2 (2) G26 containing tRNA(Phe) retain proper three-dimensional fold through tertiary interactions. Single-point energy and molecular electrostatics potential calculation studies confirmed the structural significance of tRNAs containing m(2)G26 and m 2 (2) G26 compared to tRNA with normal G26, showing that the mono-methylated (m(2)G26) and dimethylated (m 2 (2) G26) modifications are required to provide structural stability not only in the hinge region but also in the other parts of tRNA(Phe). Thus, the present study allows us to better understand the effects of modified nucleosides and ionic environment on tRNA folding.

MD SIMULATION STUDIES TO INVESTIGATE ISO-ENERGETIC CONFORMATIONAL BEHAVIOUR OF MODIFIED NUCLEOSIDES M2G AND M22G PRESENT IN tRNA

Modified nucleic acid bases are most commonly found in tRNA. These may contain modifications from simple methylation to addition of bulky groups. Methylation of the four canonical nucleotide bases at a wide variety of positions is particularly prominent among the known modification. Methylation of N2 group of guanine is a relatively common modification in tRNA and rRNA. N2-methylguanosine (m2G) is the second most often encountered nucleoside in E. coli tRNAs. N2, N2-dimethylguanosine (m22G) is found in the majority of eukaryotic tRNAs and involved in forming base pair interactions with adjacent bases. Hence, in order to understand the structural significance of these methylated nucleic acid bases we have carried out molecular dynamics simulation to see the salvation effect. The results obtained shows iso-energetic conformational behaviors for m2G and m22G. The simulation trajectory of m2G shows regular periodical fluctuations suggesting that m2G is equally stable as either s-cis or s-trans rotamers. The two rotamers of m2G may interact canonically or non-canonically with opposite base as s-trans m2G26:C/A/U44 and s-cis m2G26:A/U44. The free rotations around the C-N bond could be the possible reason for these iso-energetic conformations. Dimethylation of G has almost no influence on base pairing with either A or U. Thus, these results reveal that modified nucleosides m2G and m22G may play an important role to prevent tRNA from adopting the unusual mitochondrial like conformation.

Identification of differentially methylated regions using streptavidin bisulfite ligand methylation enrichment (SuBLiME), a new method to enrich for methylated DNA prior to deep bisulfite genomic sequencing

We have developed a method that enriches for methylated cytosines by capturing the fraction of bisulfite-treated DNA with unconverted cytosines. The method, called streptavidin bisulfite ligand methylation enrichment (SuBLiME), involves the specific labeling (using a biotin-labeled nucleotide ligand) of methylated cytosines in bisulfite-converted DNA. This step is then followed by affinity capture, using streptavidin-coupled magnetic beads. SuBLiME is highly adaptable and can be combined with deep sequencing library generation and/or genomic complexity-reduction. In this pilot study, we enriched methylated DNA from Csp6I-cut complexity-reduced genomes of colorectal cancer cell lines (HCT-116, HT-29 and SW-480) and normal blood leukocytes with the aim of discovering colorectal cancer biomarkers. Enriched libraries were sequenced with SOLiD-3 technology. In pairwise comparisons, we scored a total of 1,769 gene loci and 33 miRNA loci as differentially methylated between the cell lines and leukocytes. Of these, 516 loci were differently methylated in at least two promoter-proximal CpG sites over two discrete Csp6I fragments. Identified methylated gene loci were associated with anatomical development, differentiation and cell signaling. The data correlated with good agreement to a number of published colorectal cancer DNA methylation biomarkers and genomic data sets. SuBLiME is effective in the enrichment of methylated nucleic acid and in the detection of known and novel biomarkers.

Iron-catalysed oxidation intermediates captured in a DNA repair dioxygenase

Mononuclear iron-containing oxygenases conduct a diverse variety of oxidation functions in biology1,2, including the oxidative demethylation of methylated nucleic acids and histones3,4. E. coli AlkB is the first such enzyme that was discovered to repair methylated nucleic acids (Fig. 1)5,6, which are otherwise cytotoxic and/or mutagenic. AlkB human homologues are known to play pivotal roles in various processes7–11. Presented here is the first structural characterization of oxidation intermediates for these demethylases. Employing a chemical cross-linking strategy12,13, complexes of AlkB-dsDNA containing 1,N6-etheno adenine (εA), N3-methyl thymine (3-meT), and N3-methyl cytosine (3-meC) were stabilized and crystallized, respectively. Exposing these crystals, grown under anaerobic conditions containing iron(II) and α-ketoglutarate (αKG), to dioxygen initiates oxidation in crystallo (Supplementary Fig. 1). A glycol (from εA) and a hemiaminal (from 3-meT) intermediates are captured; a zwitterionic intermediate (from 3-2 meC) is also proposed, based on crystallographic observations and computational analysis. The observation of these unprecedented intermediates provides direct support for the oxidative demethylation mechanism for these demethylases. This study also depicts a general mechanistic view of how a methyl group is oxidatively removed from different biological substrates.

Potential Energy and Free Energy Surfaces of All Ten Canonical and Methylated Nucleic Acid Base Pairs: Molecular Dynamics and Quantum Chemical ab Initio Studies

The potential energy surfaces (PESs) and free energy surfaces (FESs) of all 10 canonical and methylated nucleic acid base pairs were studied by a molecular dynamics/quenching (MD/Q) technique with the Cornell et al. empirical force field and by a correlated ab initio quantum chemical method. More than a dozen energy minima were located on the PES of each base pair. The global and first local minima of nonmethylated base pairs have a systematically planar H-bonded structure, while T-shaped and stacked structures are less stable. The MD/Q search sometimes reveals an unexpected structure as the global energy minimum (e.g., the global minimum of the adenine...thymine PES corresponds neither to the Watson−Crick nor the Hoogsteen type of bonding). Entropy does not play an important role and the relative order of individual structures on the PES and FES does not differ too much. Methylation at purine N9 and pyrimidine N1 brings dramatic changes in the PESs and FESs, mainly because the most stable and most popula...

At nonzero temperatures, stacked structures of methylated nucleic acid base pairs and microhydrated nonmethylated nucleic acid base pairs are favored over planar hydrogen-bonded structures: a molecular dynamics simulations study.

The dynamic structure of all ten possible nucleic acid (NA) base pairs and methylated NA base pairs hydrated by a small number of water molecules (from 1 to 16) was determined by using molecular dynamics simulations in the NVE microcanonical and NVT canonical ensembles with the Cornell force field (W. D. Cornell, P. Cieplak, C. I. Bayly, I. R. Gould, K. M. Merz, D. M. Ferguson, D. C. Spellmeyer, T. Fox, J. E. Caldwell, P. Kollman, J. Am. Chem. Soc. 1995, 117, 5179). The presence of one water molecule does not affect the structure of any hydrogen-bonded (H-bonded) nonmethylated base pair. An equal population of H-bonded and stacked structures of adenine...adenine, adenine...guanine and adenine... thymine pairs is reached if as few as two water molecules are present, while obtaining equal populations of these structures in the case of adenine...cytosine, cytosine...thymine, guanine... guanine and guanine...thymine required the presence of four water molecules, and in the case of guanine...cytosine, six. A comparable population of planar, H-bonded and stacked structures for cytosine...cytosine and thymine... thymine base pairs was only obtained if at least eight water molecules hydrated a pair. Methylation of bases changed the situation dramatically and stacked structures were favoured over H-bonded ones even in the absence of water molecules in most cases. Only in the case of methyl cytosine...methyl cytosine, methyl guanine...methyl guanine and methyl guanine...methyl cytosine pairs were two, two or six water molecules, respectively, needed in order to obtain a comparable population of planar, H-bonded and stacked structures. We believe that these results give clear evidence that the preferred stacked structure of NA base pairs in the microhydrated environment, and also apparently in a regular solvent, is due to the hydrophilic interaction of a small number of water molecules. In the case of methylated bases, it is also due to the fact that the hydrogen atoms most suitable for the formation of H-bonds have been replaced by a methyl group. A preferred stacked structure is, thus, not due to a hydrophobic interaction between a large bulk of water molecules and the base pair, as believed.

Mass spectrometric and computational study of complex formation of nucleic acid bases with acrylamide as a surrogate for asparagine and glutamine residues

In the context of the problem of nucleic acid–protein interactions, thermodynamic and geometrical parameters of complexes of methylated nucleic acid bases with acrylamide (as a model of the side chains of asparagine and glutamine) were investigated. Enthalpies of formation of nucleic acid base–acrylamide complexes, determined experimentally by means of temperature-dependent field ionization mass spectrometry, were in reasonable agreement with the interaction energies calculated theoretically using atom–atom potential functions. A stability order for base–acrylamide complexes was determined: m9Gua ≥ m21,4 Cyt ≥ m1 Cyt > m9 Ade > m31,4,4 Cyt > m1 Ura > m21,3 Thy.The geometries of the most energetically favourable complexes are determined and discussed. © 1998 John Wiley & Sons, Ltd.

Multiple site proton affinities of methylated nucleic acid bases

Proton affinities of nucleic acid base molecules methylated at the site where the sugar connects to the base in the DNA helix are calculated by the use of ab initio methods. Geometry optimizations and frequency calculations are performed at the HF/6-31G(d) level, while the final energies and proton affinities are calculated with the MP2/6-311(2d,2p) method. The proton affinities are reported for temperatures of 0 and 298.15 K. The proton affinity values for the most basic protonation sites are: 891.5 kJ/mol for 1-methyluracil, 982.8 kJ/mol for 1-methylcytosine, 961.3 kJ/mol for 9-methyladenine and 980.5 kJ/mol for 9-methylguanine. The calculated proton affinity values of the singly methylated nucleic acid bases are in agreement with the experimental values for the sugarylated bases, the nucleosides.

Hydration free energies of nucleic acid bases using an ab initio continuum model

The self-consistent reaction field model, implemented within an ab initio MO framework, predicts the relative solvation energies of four methylated nucleic acid bases in close agreement with OPLS FEP calculations, in contrast to AMBER FEP results and the predictions of the semi-empirical AM1-SM2 method.

Polarization of the nucleic acid bases in aqueous solution

We present calculations of the absolute free energy of solvation of five nucleic acid bases and five methylated nucleic acid bases using a recently developed local-field SCF procedure in which the electronic structure and geometry are both optimized in the presence of solvent. The calculated solvation free energies are increased 23%–24% by the aqueous-phase relaxation.

C-H ... O hydrogen bonding in solutions of methylated nucleic acid base analogs as revealed by NMR.

Formation and thermodynamic characteristics of C-H ... O hydrogen bonding of methylated uracils and caffeine have been studied by nmr along two lines. 1. The concentration and temperature dependencies of the PMR spectra of 1,3-dimethyluracil (m2 1,3Ura), 1,3-dimethylthymine (m2 1,3Thy), and 1,3,6-trimethyluracil (m3 1,3,6Ura) in chloroform at high concentrations of base analogs indicated the self-association of m2 1,3Ura and m2 1,3Thy via C(6)H ... O hydrogen bonding and the competitive formation of C-H ... O bonds between carbonyl oxygens and chloroform. The intermolecular interaction energy and the arrangement of molecules in the local minima of various m2 1,3Ura dimers were calculated by the method of atom-atom potentials. The deepest minimum for the m2 1,3Ura coplanar dimer corresponds to a C(6)-H ... O hydrogen-bond formation. 2. At low concentration of m2 1,3Ura and caffeine in CCl4, C(6)-H ... O bonding for m2 1,3Ura and C(8)-H ... O bonding for caffeine with oxygens of dimethyl sulfoxide (DMSO) and acetone were observed. The association constants of these complexes were obtained at different temperatures. The enthalpies delta H, of the m2 1,3Ura-DMSO, m2 1,3Ura-accetone, caffeine-DMSO, and caffeine-acetone complexes were -2 +/- 0.1 kcal/mol. The calculations showed that the deepest minimum of the caffeine-acetone coplanar complex corresponds to C(8)-H ... O bonding with energy of -3.5 kcal/mol and that of the m2 1,3Ura-acetone complexes corresponds to C(6)-H ... O bonding with energy of -3.4 kcal/mol. The approximate correction for the solvent effect provides good agreement of the experimental data with the calculations.

DNA and protein adducts as indicators of in vivo methylation by nitrosatable drugs.

Exposure to methylating carcinogens may be monitored by measuring both the formation of S-methylcysteine in haemoglobin and the urinary excretion of N-7-methylguanine (7-MeG), which is derived in part from methylated nucleic acids. Female rats were exposed to methylmethanesulphonate, methylnitrosourea and to three drugs, aminopyrine, cimetidine and pyrilamine, which are potential methylating agents if nitrosation occurs in vivo. Because S-methylcysteine in haemoglobin and urinary 7-MeG occur naturally, the experiments were carried out with stable isotope-labelled analogues which contained trideutero (d3)-methyl groups. Gas chromatography-mass spectrometry was used for the quantitative determination of d3-labelled adducts after their separation from the biological matrix and chemical derivatization. Transfer of the intact d3-methyl group to cysteine and guanine was detected after intragastric administration of d3-methyl-methanesulphonate (50 mg/kg), d3-N-methyl-N-nitrosourea (50 mg/kg), and d6-aminopyrine (AP) and sodium nitrite (both 100 mg/kg). AP alone gave no detectable d3-methyl adducts. Co-administration of nitrite and d6-pyrilamine or d3-cimetidine yielded no d3-7-MeG, although N-nitroso-d3-cimetidine alkylated DNA in vitro in a dose-dependent fashion. For AP and nitrite combinations urinary excretion of d3-7-MeG was linearly related to the dose of nitrite and was essentially complete within 5 days. For d3-methylmethane-sulphonate (50 mg/kg) the ratio of haemoglobin d3-S-methylcysteine to urinary d3-7-MeG was considerably (greater than 9-fold) higher than for either d3-N-methyl-N-nitrosourea or AP/nitrite (100 mg/kg) mixture. This is in accord with the SN2 nature of the weak carcinogen methylmethanesulphonate compared with the SN1 nature of the reactive methylating agent derived from either one of the N-methyl-N-nitroso compounds.

Functional Monomers and Polymers CXXII. Synthesis of Polymethacrylates Containing Nucleic Acid Bases and Their Methylated Derivatives by Copolymerization

Polymethacrylate derivatives containing both nucleic acid bases and methylated nucleic acid bases were prepared by free radical copolymerizations of the corresponding monomers to study the effects of methylation of nucleic acid bases on specific interactions. The methacylate monomer containing 3-methyluracil was prepared by methylation of 1-(2-hydroxyethyl)uracil followed by a reaction with methacrylic anhydride. The free radical copolymerization of this monomer with a monomer containing uracil gave the polymethacrylate containing uracil and 3-methyl uracil units. The polymethacrylate containing adenine and N6-methyladenine units was also prepared using a similar procedure to that for the uracil derivative.

Functional Monomers and Polymers CXXIII. Interaction of Polymethacrylates Containing Nucleic Acid Bases: Effect of Methylation of Nucleic Acid Bases

A study was made of the interaction of the polymethacrylate derivatives of nucleic acid bases, particularly the effect of methylation of nucleic acid bases in the polymers. One of the complementary polymers is a copolymer containing uracil and 3-methyluracil units, and the other, a copolymer containing adenine and N6-methyladenine units. For the interaction of these copolymers, an optimum content of the methylated nucleic acid base unit in the copolymer was found. This was explained by opposite factors: an inhibition of hydrogen bonding and increase in compatibility or mutual solubility of two kinds of the copolymers.

The action of rat cytosol enzymes on some methylated nucleic acid components produced by the carcinogenic N-nitroso compounds

The stabilities of several alkylated nucleic acid components have been examined in the presence of cytosol extracts from a variety of rat tissues. An activity capable of demethylating O 6-methyldeoxyguanosine was readily detectable in all tissues examined; arranged in approximate order of decreasing specific activity these are as follows: small intestine, spleen, kidney, lung, liver, skin, heart and brain. The in vitro requirements for the activity derived from liver and the observations that O 6-methylguanine and its deoxynucleoside 5'-monophosphate are insensitive to the action of these extracts suggests that this activity may be due to an enzyme which resembles adenosine deaminase. In contrast to the ready degradation of O 6-methyldeoxyguanosine the corresponding ethyl derivative was degraded very much more slowly but there was no evidence for other activities against the O 4-andO 2-methyldeoxythymidines. Similarly, no demethylation of the N-substituted deoxynucleosides, 3-methyldeoxycytidine 3-methyldeoxythymidine, 1-methyldeoxyadenosine and 7-methyldeoxyguanosine, was detected.