Widespread existence of Cytosine methylation in yeast DNA measured by gas chromatography/mass spectrometry

DNA methylation is one of the major epigenetic modifications and has been involved in a number of biological processes in mammalian cells. Yeast is widely used as a model organism for studying cell metabolism, cell cycle regulation, and signal transduction. However, it remains controversial whether methylated cytosine (5-methylcytosine, 5mC) exists in the yeast genome. In the current study, we developed a highly sensitive method based on gas chromatography/mass spectrometry (GC/MS) and systematically examined the incidence of 5mC in 19 yeast strains, which represent 16 yeast species. Our results showed that DNA methylation is widespread in yeast and the genome-wide DNA methylation of the studied yeast strains ranged from 0.014 to 0.364%, which were 1 to 2 orders of magnitude lower than that in mammalian cells (i.e., 3-8%). Furthermore, we found that the 5mC content in yeast varied considerably at different growth stages and DNA methylation inhibitor 5-azacytidine could induce a decrease in genome-wide DNA methylation as that in mammalian cells. The demonstration of the universal presence of DNA cytosine methylation in yeast constituted the first and essential step toward understanding the functions of this methylation in yeast.     

Electrochemical DNA methylation detection for enzymatically digested CpG oligonucleotides

We describe the electrochemical detection of DNA methylation through the direct oxidation of both 5-methylcytosine (mC) and cytosine (C) in 5'-CG-3' sequence (CpG) oligonucleotides using a sputtered nanocarbon film electrode after digesting a longer CpG oligonucleotide with endonuclease P1. Direct electrochemistry of the longer CpG oligonucleotides was insufficient for obtaining the oxidation currents of these bases because the CG rich sequence inhibited the direct oxidation of each base in the longer CpG oligonucleotides, owing to the conformational structure and its very low diffusion coefficient. To detect C methylation with better quantitativity and sensitivity in the relatively long CpG oligonucleotides, we successfully used an endonuclease P1 to digest the target CpG oligonucleotide and yield an identical mononucleotide 2'-deoxyribonucleoside 5'-monophosphate (5'-dNMP). Compared with results obtained without P1 treatment, we achieved 4.4 times higher sensitivity and a wider concentration range for mC detection with a resolution capable of detecting a subtle methylated cytosine difference in the CpG oligonucleotides (60mer).     

Fluorescently imaged particle counting immunoassay for sensitive detection of DNA modifications

Modifications of genomic DNA may change gene expression and cause adverse health effects. Here we for the first time demonstrate a particle counting immunoassay for rapid and sensitive detection of DNA modifications using benzo[a]pyrenediol epoxide (BPDE)-DNA adducts as an example. The BPDE-adducted DNA is specifically captured by immunomagnetic particles and then isolated from unmodified DNA by applying an external magnetic field. By taking advantage of the fluorescence signal amplification through multiple labeling of captured DNA by OliGreen dye, the captured BPDE-DNA adducts can be quantified by particle counting from fluorescence imaging. This clearly demonstrates that the number of fluorescently countable particles is proportional to the modification content in genomic DNA. It is interesting to note that the background fluorescence signal caused by nonspecific adsorption of OliGreen dye can be more effectively quenched than that induced by the binding of OliGreen dye to ssDNA, allowing for significant reduction in the background fluorescence and further enhancing the detection sensitivity. The developed method can detect trace BPDE-DNA adducts as low as 180 fM in the presence of 1 billion times more normal nucleotides in genomic DNA and has a dynamic range over 4 orders of magnitude. By using anti-5-methylcytosine antibody, the method is extended to the detection of global DNA methylation. With high sensitivity and specificity, this rapid and easy-to-perform analytical method for DNA modifications shows a broad spectrum of potential applications in genotoxical and epigenetic analysis.     

Theoretical investigation of interaction between Gatifloxacin and DNA: Implications for anticancer drug design

This study is an attempt to have a better understanding of the physicochemical interaction between a novel anticancer drug, Gatifloxacin (GTFX) and its biological receptor, DNA. The eventual purpose is to design drugs which interact more with DNA. Considering the physicochemical properties of the drug as well as the mechanism by which it interacts with DNA, it should eventually allow the rational design of novel anticancer or antiviral drugs. Molecular modeling on the complex formed between Gatifloxacin and DNA presented the full ability of the drug for participating in the formation of a stable intercalation site. Besides, the molecular geometries of Gatifloxacin (GTFX) and the DNA bases (Adenine, Guanine, Cytosine and Thymine) were optimized with the aid of B3LYP/6-31G? method. The properties of the isolated intercalator and its stacking interactions with the adenine?thymine (AT) and guanine?cytosine (GC) nucleic acid base pairs, were studied using DFTB method, (an approximate version of the DFT method) that was extended to cover the London dispersion energy. The B3LYP/6-31G? stabilization energies of the intercalator?base pair complexes were found to be ? 26.99 and ? 37.62 kcal/mol for AT?GTFX and GC?GTFX, respectively. It was completed that the dispersion energy and the electrostatic interaction contributed to the stability of the intercalator?DNA base pair complexes.     

Nanomechanical recognition measurements of individual DNA molecules reveal epigenetic methylation patterns

Atomic force microscopy (AFM) is a powerful tool for analysing the shapes of individual molecules and the forces acting on them. AFM-based force spectroscopy provides insights into the structural and energetic dynamics of biomolecules by probing the interactions within individual molecules, or between a surface-bound molecule and a cantilever that carries a complementary binding partner. Here, we show that an AFM cantilever with an antibody tether can measure the distances between 5-methylcytidine bases in individual DNA strands with a resolution of 4 ?, thereby revealing the DNA methylation pattern, which has an important role in the epigenetic control of gene expression. The antibody is able to bind two 5-methylcytidine bases of a surface-immobilized DNA strand, and retracting the cantilever results in a unique rupture signature reflecting the spacing between two tagged bases. This nanomechanical approach might also allow related chemical patterns to be retrieved from biopolymers at the single-molecule level.     

"Multicolor" electrochemical labeling of DNA hybridization probes with osmium tetroxide complexes

Labeling of oligonucleotide reporter probes (RP) with electroactive markers has frequently been utilized in electrochemical detection of DNA hybridization. Osmium tetroxide complexes with tertiary amines (Os,L) bind covalently to pyrimidine (predominantly thymine) bases in DNA, forming stable, electrochemically active adducts. We propose a technique of electrochemical "multicolor" DNA coding based on RP labeling with Os,L markers involving different nitrogenous ligands (such as 2,2' -bipyridine, 1,10-phenanthroline derivatives or N,N,N',N'-tetramethylethylenediamine). At carbon electrodes the Os,L-labeled RPs produce specific signals, with the potentials of these differing depending on the ligand type. When using Os,L markers providing sufficiently large differences in their peak potentials, parallel analysis of multiple target DNA sequences can easily be performed via DNA hybridization at magnetic beads followed by voltammetric detection at carbon electrodes. Os,L labeling of oligonucleotide probes comprising a segment complementary to target DNA and an oligo(T) tail (to be modified with the osmium complex) does not require any organic chemistry facilities and can be achieved in any molecular biological laboratory. We also for the first time show that this technology can be used for labeling of oligonucleotide probes hybridizing with target DNAs that contain both purine and pyrimidine bases.     

Interaction of chemically modified antisense oligonucleotides with sense DNA: a label-free interaction study with reflectometric interference spectroscopy.

Antisense oligonucleotides (ON) are regarded as potential therapeutic agents for controlling gene expression at the mRNA level. The strength of the interaction with the target sequence is one critical factor for the therapeutic efficiency of an ON. Herein, the results of studies on antisense 15mer and 20mer ONs against mdr1b-mRNA are described. The mdr1b is a member of the group that encodes the P-glycoprotein (Pgp), responsible for the phenomenon of multidrug resistance. The effects of backbone modification (DNA, phosphorothioate (PTO)), terminal modifications (hexadecyl, cholesteryl, tocopherol, polyethylenglycol, 2'-O-methyl-modified RNA) and base sequence misalignments (1 to 3 bases) on interaction kinetics and binding strength were investigated. The interaction of an immobilized sense strand with the dissolved antisense ON was monitored with a label-free optical transducer based on thin film interference (RIfS). Association kinetics were detected at a low density of immobilized ON. Thermodynamics were investigated by homogeneous phase titration of sense and antisense ON and subsequent quantification of equilibrium concentrations of unbound ON at a transducer highly loaded with sense ON. Association rate constants varied from 3.1 (+/- 0.2) x 10(4) M-1 s-1 (poly(ethylene glycol)-modified DNA strand) to 4.3 (+/- 0.1) x 10(4) M-1 s-1 (hexadecyl-modified strand). Binding constants varied from 1.9 (+/- 0.1) x 10(8) M-1 (cholesteryl modification) to 5 (+/- 0.4) x 10(7) M-1 (tocopherol modification). Phosphorothioate ON showed a reduction in binding strength of more than 1 order of magnitude. The data presented give valuable information for the efficiency of modified antisense oligonucleotides.     

Prediction of collision-induced-dissociation spectra of peptides with post-translational or process-induced modifications

Mass spectrometry, combined with collision-induced dissociation (CID), has become the method of choice for analyzing protein post-translational and process-induced modifications. However, confident and automated identification of modifications and modification sites is often challenged by the diversity of modifications and their labile nature under typical CID conditions. An accurate prediction of the CID spectra of modified peptides will improve the reliability of automated determination of modifications and modification sites. In this article, the kinetic model for the prediction of peptide CID spectra is extended to the prediction of the CID spectra of modified peptides. The mathematical model for predicting CID spectra of peptides with enzymatic and chemical modifications such as (1) phosphorylation of serine, threonine, and tyrosine, (2) S-carboxymethylation and carbamidomethylation of cysteine, (3) different stages of oxidation of methionine, tryptophan, and cysteine, (4) glycation of lysine, (5) O-mannosylation of serine, (6) hydroxylation of lysine, and (7) N-monomethylation and N-dimethylation of lysine is described. The mathematical model, once established with CID spectra of peptides with known modifications and modification sites, is able to predict CID spectra with excellent accuracy in ion intensities, facilitating more reliable identification of modification and modification sites.     

Benzo-homologated nucleobases in a nanotube-electrode set-up for DNA sequencing

Motivated by the possibility that the conductivity signatures of benzo-homologated DNA bases could be used to sequence DNA, we have investigated the conductivity properties of these bases when they are non-covalently sandwiched between two (5,5) nanotube electrodes. It is found that these bases conduct poorly, making it very difficult to distinguish them. An analysis of the changes in the conductivity of benzo-adenine as a function of the distance between the tips of the nanotubes revealed that, even though the conductance increases by four orders of magnitude when the electrodes are brought closer together, the net conductance remains rather small. These results suggest that benzo-homologated bases, despite having smaller HOMO-LUMO gaps than their natural counterparts, when non-covalently bound to the electrodes cannot be used to sequence DNA by means of conductivity measurements.     

Atomistic view of base flipping in DNA

Base flipping is essential for the enzyme-catalysed methylation of DNA. In our previous studies, the flipping of bases out of duplex DNA in DNA alone and when bound to the (cytosine-C5)-methyltransferase from HhaI (M.HhaI) were investigated via potential of mean force calculations. Insights into various experimental observations were obtained. In the present paper we present an overview of previous computational studies of base flipping along with new detailed structural and energetic analysis on atomic events that contribute to the free energy surfaces. The contributions from different intrinsic and environmental effects to the base-flipping process are explored, and experimental data derived from a variety of methods are reconciled. A detailed protein-facilitated base-flipping mechanism is proposed. Ground-state destabilization is achieved via disruption of the target base Watson-Crick interactions by substitution with favourable DNA-protein interactions. In addition, specific DNA-protein interactions and favourable solvation effects further promote target base flipping along the major groove through the protein matrix, and maximal interactions occur between the DNA and the protein upon reaching the fully flipped state. Other DNA binding proteins that involve distortion of DNA's conformation may use a similar mechanism to that by which M.HhaI facilitates base flipping.     

Complexes of DNA bases and gold clusters Au3 and Au4 involving nonconventional N-H...Au hydrogen bonding

DNA base-gold interactions are studied theoretically at the DFT level using Au(3) and Au(4) clusters as simple catalytic models for Au particles. The bonding between DNA bases and gold clusters occurs via the anchoring of a Au atom to the N or O atoms of the bases. In the most stable planar base-Au(3) complexes, the Au-N or Au-O anchor bonds are reinforced by N-H...Au bonds. The mechanism of formation of these nonconventional H-bonds is discussed.     

A Molecular Hero Suit for In Vitro and In Vivo DNA Nanostructures

Precise control of DNA base pairing has rapidly developed into a field full of diverse nanoscale structures and devices that are capable of automation, performing molecular analyses, mimicking enzymatic cascades, biosensing, and delivering drugs. This DNA‐based platform has shown the potential of offering novel therapeutics and biomolecular analysis but will ultimately require clever modification to enrich or achieve the needed “properties” and make it whole. These modifications total what are categorized as the molecular hero suit of DNA nanotechnology. Like a hero, DNA nanostructures have the ability to put on a suit equipped with honing mechanisms, molecular flares, encapsulated cargoes, a protective body armor, and an evasive stealth mode.     

共混改性提高奇-奇数尼龙1111的压电与铁电性能

压电与铁电聚合物在换能器、驱动器与传感器中应用广泛,但是铁电聚合物种类稀少,而尼龙类铁电聚合物具有廉价、绿色以及高温稳定的特点,有潜在的应用价值。奇-奇数尼龙1111(PA1111)是一种新型铁电与压电聚合物材料,但其电活性偏低,限制了它的应用。通过与聚偏氟乙烯(PVDF)共混改性进一步提高了PA1111的电活性。研究表明,添加40%PVDF的尼龙1111拉伸薄膜压电应变常数(d33)提高到-6.9 pC/N,剩余极化强度(Pr)增加到52 mC/m^2。共混改性后PA1111铁电与压电性能的提高可以归因于PA1111与PVDF偶极子间的相互作用。     

DNA capillary electrophoresis in entangled dynamic polymers of surfactant molecules

Aqueous solutions of monomeric nonionic surfactants, n-alkyl polyoxyethylene ethers (C16E6, C16E8, C14E6), can be used as sieving matrixes for the separation of DNA fragments by capillary electrophoresis. Unlike ordinary polymer solutions, these surfactant solutions behave as dynamic polymers. By combining the "reversible gel" theory of DNA electrophoresis and the static and dynamic properties of wormlike surfactant micelles, a model is developed for describing the migration behavior of DNA molecules in these solutions. According to the model, the separation limit can be extended at low surfactant concentrations. Surfactant solutions as a separation medium provide many advantages over ordinary polymers, such as ease of preparation, solution homogeneity, stable structure, low viscosity, and self-coating property for reducing electroosmotic flow. More importantly, the properties of wormlike micelles (micelle size, entanglement concentration) can be adjusted by simply changing the monomer concentration, denaturant, and temperature to allow the separation of different size ranges of DNA fragments. Fast separation is achieved for DNA fragments ranging from 10 bp to 5 kb by using bare fused-silica columns. DNA sequencing fragments of BigDye G-labeled M13 up to 600 bases were separated within 60 min.     

Identifying DNA methylation in a nanochannel

DNA methylation is a stable epigenetic modification, which is well known to be involved in gene expression regulation. In general, however, analyzing DNA methylation requires rather time consuming processes (24–96 h) via DNA replication and protein modification. Here we demonstrate a methodology to analyze DNA methylation at a single DNA molecule level without any protein modifications by measuring the contracted length and relaxation time of DNA within a nanochannel. Our methodology is based on the fact that methylation makes DNA molecules stiffer, resulting in a longer contracted length and a longer relaxation time (a slower contraction rate). The present methodology offers a promising way to identify DNA methylation without any protein modification at a single DNA molecule level within 2 h.     

Use of DNA repair enzymes in electrochemical detection of damage to DNA bases in vitro and in cells

Electrochemical measurements at mercury or solid amalgam electrodes offer a highly sensitive detection of DNA strand breaks. On the other hand, electrochemical detection of damage to DNA bases at any electrode is usually much less sensitive. In this paper, we propose a new voltammetric method for the detection of the DNA base damage based on enzymatic conversion of the damaged DNA bases to single-strand breaks (ssb), single-stranded (ss) DNA regions, or both. Supercoiled DNA exposed to UV light was specifically cleaved by T4 endonuclease V, an enzyme recognizing pyrimidine dimers, the major products of photochemical DNA damage. Apurinic sites (formed in dimethyl sulfate-modified DNA) were determined after treating the DNA with E. coli exonuclease III, an enzyme introducing ssb at the abasic sites and degrading one of the DNA strands. The ssb or ssDNA regions, or both, were detected by adsorptive transfer stripping alternating current voltammetry at the mercury electrode. This technique offers much better sensitivity and selectivity of DNA base damage detection than any other electrochemical method. It is not limited to DNA damage in vitro, but it can detect also DNA base damage induced in living bacterial cells.     

Chemical aspects of clustered DNA damage induction by ionising radiation

Ionising radiation induces a variety of chemical modifications to DNA, ranging from simple, isolated lesions to clustered DNA damage, in which two or more lesions are formed within a few tens of base pairs by a single radiation track. Multiple lesions, e.g. tandem lesions and amplification of damage, may also be induced in DNA by reaction with a single hydroxyl radical. It has been proposed from biophysical modelling that clustered DNA damage is less repairable and therefore contributes to the biological severity of ionising radiation. In this review, some evidence is presented which indicates that non-double strand break (non-DSB) clustered DNA damage is induced in significant yield, relative to that of DSBs, in mammalian cells. Enzymatic processing of clustered DNA damage in synthetic oligonucleotides has been shown to be compromised, depending on the nature of the lesions present. The role of clustered DNA damage in the early stages of the development of radiation-induced carcinogenesis remains to be addressed.     

Sensitive detection of intracellular sumoylation via SNAP tag-mediated translation and RNA polymerase-based amplification

The covalent attachment of small ubiquitin-like modifier (SUMO) to target proteins, defined as sumoylation, is an important post-translational modification that regulates diverse cellular processes and many human diseases. However, functional analysis of sumo modification is usually hampered by the lack of sensitive methods for measuring extremely low abundance of specific sumoylated target in the cells. Here, we develop an ultrasensitive method for intracellular sumoylation assay based on SNAP tag (a mutant of O6-alkylguanine-DNA alkyltransferase)-mediated translation and RNA polymerase-based amplification. Intracellular sumo modification is first converted to the double-stranded DNA (dsDNA) containing the specific T7 promoter sequence via the covalent conjugation of SNAP tag with its substrate benzyl guanidine derivate; then, the dsDNA is extensively transcribed by T7 RNA polymerase to produce large amounts of RNAs, which are easily monitored using the RNA intercalating dye RiboGreen and a standard fluorometer. This method exhibits excellent specificity and high sensitivity and can detect as little as 5 pg of sumoylated p53 proteins, which has improved by as much as 1000-fold than that in the conventional Western blotting assay. Moreover, this method can measure intracellular sumoylation under different physiological conditions. Due to the common translation and amplification module, this method can be further extended to detect a variety of sumoylated proteins and other ubiquitin-like modifications in the cells and might provide a powerful tool for comprehensive analysis of the functions of sumoylation and other ubiquitin-like modifications in the fundamental biological processes and many human diseases.     

A highly efficient fluorescence-based switch-on detection method of 5-formyluracil in DNA

The identification of hydroxylmethyl-and formylpyrimidines in genomic DNA was a landmark event in epigenetics.Numerous laboratories in related fields are investigating the biology of these and other nucleic acid modifications.However,limitations in the ability to detect and synthesize appropriate modifications are an impediment.Herein,we explored a remarkable development in the selective detection of 5-formyluracil in both single-stranded and double-stranded DNA under mild conditions.The "switch-on" specificity towards 5-formyluracil enabled a high signal-to-noise ratio in qualitatively and quantitatively detecting materials containing 5-forrnyluracil,which is not affected by the presence of abasic sites and 5-formylcytosine,the modified cytosine counterpart of 5-formyluracil.In summar~ the innoxiousness,convenience,and cost-efficiency of the 5-formyluracil phosphoramidite synthetic routine would promote the understanding of the epigenetic role of this natural thymidine modification.     

A method to assess genomic DNA methylation using high-performance liquid chromatography/electrospray ionization mass spectrometry

Eukaryotic DNA is methylated at some cytosine residues, and this epigenetic feature performs critical functions. We developed a method for quantitative determination of 5-methyl-2'-deoxycytidine in human DNA using liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). The DNA was enzymatically hydrolyzed by sequential digestion with three enzymes. DNA hydrolyzates were subsequently separated by reversed-phase high-performance liquid chromatography in isocratic mode. The four major DNA bases and 5-methyl-2'-deoxycytidine were resolved and eluted in 13 min. Identification of 2'-deoxycytidine and 5-methyl-2'-deoxycytidine was obtained by combined diode array UV spectra analysis and mass spectra of chromatographic peaks. The isotopomers [15N3]-2'-deoxycytidine and (methyl-d3,ring-6-d1)-5-methyl-2'-deoxycytidine were used as internal standards. Ions of m/z 126 and 130 were used to detect 5-methyl-2'-deoxycytidine and its isotopomer, and ions of m/z 112 and 115 were used to detect 2'-deoxycytidine and its stable isotopomer, respectively. The DNA methylation status was calculated on the basis of the amount of 5-methyl-2'-deoxycytidine per microgram of DNA with percent relative standard deviations (%RSD) for a method precision of 7.1 (within-day) and 5.7 (day-to-day). This method also allows the measurement of 5-methyl-2'-deoxycytidine expressed as a percentage of total deoxycytidine residues in genomic DNA with %RSD for method precision of 1.9 (within-day) and 1.7 (day-to-day). This LC/MS method for quantitative determination of genomic DNA methylation status is rapid, sensitive, selective, and precise.