Cytosine Sequence Research Articles

GCparagon: evaluating and correcting GC biases in cell-free DNA at the fragment level.

Analyses of cell-free DNA (cfDNA) are increasingly being employed for various diagnostic and research applications. Many technologies aim to increase resolution, e.g. for detecting early-stage cancer or minimal residual disease. However, these efforts may be confounded by inherent base composition biases of cfDNA, specifically the over - and underrepresentation of guanine (G) and cytosine (C) sequences. Currently, there is no universally applicable tool to correct these effects on sequencing read-level data. Here, we present GCparagon, a two-stage algorithm for computing and correcting GC biases in cfDNA samples. In the initial step, length and GC base count parameters are determined. Here, our algorithm minimizes the inclusion of known problematic genomic regions, such as low-mappability regions, in its calculations. In the second step, GCparagon computes weights counterbalancing the distortion of cfDNA attributes (correction matrix). These fragment weights are added to a binary alignment map (BAM) file as alignment tags for individual reads. The GC correction matrix or the tagged BAM file can be used for downstream analyses. Parallel computing allows for a GC bias estimation below 1 min. We demonstrate that GCparagon vastly improves the analysis of regulatory regions, which frequently show specific GC composition patterns and will contribute to standardized cfDNA applications.

Signal-On and Highly Sensitive Electrochemiluminescence Biosensor for Hydrogen Sulfide in Joint Fluid Based on Silver-Ion-Mediated Base Pairs and Hybridization Chain Reaction

Hydrogen sulfide (H2S) in joint fluid acts as a signal molecule to regulate joint inflammation. Direct detection of H2S in joint fluid is of great significance for the diagnosis and treatment of arthritis. However, due to the low volume of joint fluid and low H2S concentration, existing methods face the problem of the insufficient limit of detection. In this study, a highly sensitive biosensor was proposed by designing a primer probe and combining it with hybrid chain reaction (HCR) under the strong interaction between metal ions and H2S to achieve H2S detection. The primer probe containing multiple cytosine (C) sequences was fixed on a gold electrode, and the C–Ag–C hairpin structure was formed under the action of Ag+. In the presence of H2S, it can combine with Ag+ in the hairpin structure to form Ag2S, which leads to the opening of the hairpin structure and triggers the hybridization chain reaction (HCR) with another two hairpin structures (H1 and H2). A large number of double-stranded nucleic acid structures can be obtained on the electrode surface. Finally, Ru(phen)32+ can be embedded into the double chain structure to generate the electrochemiluminescence (ECL) signal. The linear response of the H2S biosensor ranged from 0.1000 to 1500 nM, and the limit of detection concentration of H2S was 0.0398 nM. The developed biosensor was successfully used to determine H2S in joint fluid.

Integrated analysis of DNA methylome and transcriptome reveals epigenetic regulation of CAM photosynthesis in pineapple

BackgroundCrassulacean acid metabolism (CAM) photosynthesis is an important carbon fixation pathway especially in arid environments because it leads to higher water-use efficiency compared to C3 and C4 plants. However, the role of DNA methylation in regulation CAM photosynthesis is not fully understood.ResultsHere, we performed temporal DNA methylome and transcriptome analysis of non-photosynthetic (white base) and photosynthetic (green tip) tissues of pineapple leaf. The DNA methylation patterns and levels in these two tissues were generally similar for the CG and CHG cytosine sequence contexts. However, CHH methylation was reduced in white base leaf tissue compared with green tip tissue across diel time course in both gene and transposon regions. We identified thousands of local differentially methylated regions (DMRs) between green tip and white base at different diel periods. We also showed that thousands of genes that overlapped with DMRs were differentially expressed between white base and green tip leaf tissue across diel time course, including several important CAM pathway-related genes, such as beta-CA, PEPC, PPCK, and MDH.ConclusionsTogether, these detailed DNA methylome and transcriptome maps provide insight into DNA methylation changes and enhance our understanding of the relationships between DNA methylation and CAM photosynthesis.

C-Ag+−C based repetitive DNA sequence

DNA is a convenient and well-studied tool for nanostructures fabrication. Metal-mediated hybridization of DNA strands opens up new possibilities for nanobiotechnology. In this work, we studied the possibility of long DNA formation from short ones by gluing them through the formation of C-Ag+−C complexes. Such long formations were investigated using static light scattering and atomic force microscopy. It was found that the duplexes can efficiently be linked in the presence of silver ions if the length of the cytosine sequence exceeds 6 nucleobases.

In silico structural analysis of sequences containing 5-hydroxymethylcytosine reveals its potential as binding regulator for development, ageing and cancer-related transcription factors

ABSTRACT The presence of 5-hydroxymethyl cytosine in DNA has been previously associated with ageing. Using in silico analysis of normal liver samples we presently observed that in 5-hydroxymethyl cytosine sequences, DNA methylation is dependent on the co-presence of G-quadruplexes and palindromes. This association exhibits discrete patterns depending on G-quadruplex and palindrome densities. DNase-Seq data show that 5-hydroxymethyl cytosine sequences are common among liver nucleosomes (p < 2.2x10−16) and threefold more frequent than nucleosome sequences. Nucleosomes lacking palindromes and potential G-quadruplexes are rare in vivo (1%) and nucleosome occupancy potential decreases with increasing G-quadruplexes. Palindrome distribution is similar to that previously reported in nucleosomes. In low and mixed complexity sequences 5-hydroxymethyl cytosine is frequently located next to three elements: G-quadruplexes or imperfect G-quadruplexes with CpGs, or unstable hairpin loops (TCCCAY6TGGGA) mostly located in antisense strands or finally A-/T-rich segments near these motifs. The high frequencies and selective distribution of pentamer sequences (including TCCCA, TGGGA) probably indicate the positive contribution of 5-hydroxymethyl cytosine to stabilize the formation of structures unstable in the absence of this cytosine modification. Common motifs identified in all total 5-hydroxymethyl cytosine-containing sequences exhibit high homology to recognition sites of several transcription factor families: homeobox, factors involved in growth, mortality/ageing, cancer, neuronal function, vision, and reproduction. We conclude that cytosine hydroxymethylation could play a role in the recognition of sequences with G-quadruplexes/palindromes by forming epigenetically regulated DNA ‘springs’ and governing expansions or compressions recognized by different transcription factors or stabilizing nucleosomes. The balance of these epigenetic elements is lost in hepatocellular carcinoma.

Prediction of Genome Sequences in Terms of Cellular Automata Expansion of Rule Based Logics

This paper proposes a novel concept called “Percentage Nucleotide Concentration of genomes” in terms of cellular automata evolutions of adjoints of Adenine, Thymine, Guanine, and Cytosine. The adjoints of the given a genome sequenceare the characteristic binary string sequences. For example, the adjoint of Adenine of a given genome sequence is a binary string consisting of 0’s and 1’s where 1’s corresponds to the presence of Adenine in the genome sequence. So, one can have four adjoint sequences of Adenine, Thymine, Guanine, and Cytosine corresponding to a given genome sequence. One-dimensional three neighborhood binary value cellular automata rules could be applied to an adjoint sequence and the desired number of evolutions obtained.These rules aredefined by linear Boolean functions and one can have 256 such linear Boolean functions. The analysis of genome sequences with predictive analytics gives a scope of getting the inherent properties of the genome. The predictive model suits the Nucleotide concentration and is computed for an adjoint sequence and its variation evaluated for its successive evolutions based on a cellular automaton rule.

Comparative Electrochemical and Spectroscopic Studies of I‐Motif‐forming DNA Nonamers

AbstractThe paper shows the structural diversity of cytosine (C)‐rich oligodeoxynucleotides (ODNs) arising from their detail nucleotide sequence and experimental conditions. In slightly acidic solutions, the ODN nonamers with different adenine (A) and cytosine (C) sequences can adopt non‐canonical structures involving protonated bases. A distinct secondary structure formed in (C)‐rich sequences, called i‐motif (iM), consists of hemiprotonated and intercalated cytosine base pairs (C.C+). Folding and unfolding of particular structures in solutions were monitored by 1H NMR and CD spectroscopies and native polyacrylamide gel electrophoresis (PAGE), which are capable to determine their structural characteristics. Effects of sequences and their proclivity to formation of the iM on electrochemical behaviour of the ODN nonamers were studied by electrochemical methods. The LSV signals of A and C obtained from the reductive dissolution of ODN adsorption layers on a hanging mercury drop electrode were processed by elimination voltammetry with linear scan (EVLS), which revealed complex effects of the nonamer properties (namely their primary and secondary structure confirmed in solution) on their adsorption and reduction activity.

Detailed insight into the dynamics of the initial phases of de novo RNA-directed DNA methylation in plant cells

BackgroundMethylation of cytosines is an evolutionarily conserved epigenetic mark that is essential for the control of chromatin activity in many taxa. It acts mainly repressively, causing transcriptional gene silencing. In plants, de novo DNA methylation is established mainly by RNA-directed DNA-methylation pathway. Even though the protein machinery involved is relatively well-described, the course of the initial phases remains covert.ResultsWe show the first detailed description of de novo DNA-methylation dynamics. Since prevalent plant model systems do not provide the possibility to collect homogenously responding material in time series with short intervals, we developed a convenient system based on tobacco BY-2 cell lines with inducible production of siRNAs (from an RNA hairpin) guiding the methylation machinery to the CaMV 35S promoter controlling GFP reporter. These lines responded very synchronously, and a high level of promoter-specific siRNAs triggered rapid promoter methylation with the first increase observed already 12 h after the induction. The previous presence of CG methylation in the promoter did not affect the methylation dynamics. The individual cytosine contexts reacted differently. CHH methylation peaked at about 80% in 2 days and then declined, whereas CG and CHG methylation needed more time with CHG reaching practically 100% after 10 days. Spreading of methylation was only minimal outside the target region in accordance with the absence of transitive siRNAs. The low and stable proportion of 24-nt siRNAs suggested that Pol IV was not involved in the initial phases.ConclusionsOur results show that de novo DNA methylation is a rapid process initiated practically immediately with the appearance of promoter-specific siRNAs and independently of the prior presence of methylcytosines at the target locus. The methylation was precisely targeted, and its dynamics varied depending on the cytosine sequence context. The progressively increasing methylation resulted in a smooth, gradual inhibition of the promoter activity, which was entirely suppressed in 2 days.

UAP56 associates with DRM2 and is localized to chromatin in Arabidopsis.

Repeated sequence expression and transposable element mobilization are tightly controlled by multilayer processes, which include DNA 5′‐cytosine methylation. The RNA‐directed DNA methylation (RdDM) pathway, which uses siRNAs to guide sequence‐specific directed DNA methylation, emerged specifically in plants. RdDM ensures DNA methylation maintenance on asymmetric CHH sites and specifically initiates de novo methylation in all cytosine sequence contexts through the action of DRM DNA methyltransferases, of which DRM2 is the most prominent. The RdDM pathway has been well described, but how DRM2 is recruited onto DNA targets and associates with other RdDM factors remains unknown. To address these questions, we developed biochemical approaches to allow the identification of factors that may escape genetic screens, such as proteins encoded by multigenic families. Through both conventional and affinity purification of DRM2, we identified DEAD box RNA helicases U2AF56 Associated Protein 56 (UAP56a/b), which are widespread among eukaryotes, as new DRM2 partners. We have shown that, similar to DRM2 and other RdDM actors, UAP56 has chromatin‐associated protein properties. We confirmed this association both in vitro and in vivo in reproductive tissues. In addition, our experiments also suggest that UAP56 may exhibit differential distribution in cells depending on plant organ. While originally identified for its role in splicing, our study suggests that UAP56 may also have other roles, and our findings allow us to initiate discussion about its potential role in the RdDM pathway.

Microarrays with a pillared patterned double-layer graphene substrate: A molecular dynamics simulation approach

Microarray technology plays an important role in detecting the sequence of the genome. In the production of microarrays, the formation of a homogeneous and uniform spot due to biodroplet pining is of great importance. In this work, using the molecular dynamics simulation method, it is possible to create homogeneous spots on the surface of graphene-based surfaces. Hence, the behavior of five different types of DNA including homogeneous sequences of adenine, cytosine, guanine, and thymine as well as heterogeneous sequence of bases in the water droplets, was subjected to a double-layer graphene substrate with a pillar structure. The results showed that double layer graphene with pillar pattern are successful in pinning of biodroplet and this substrate leads to the differnet placement of Oligonucleotide probes. Also, the results of wettability demonstrated that the formation of hydrogen bond between a single strand and water molecules reduces the spreading and increases the contact angle and height of biodroplet containing oligonucleotides with respect to the water droplet without oligonucleotides on the surface of the double layer graphene in pillar pattern. The results of the simulation of single-stranded DNA after equilibration showed that single strand of adenine and heterogeneous single strands had the least folding compared to other single strands, and the single strand of adenine produced the most stable structure. Hence, single-stranded adenine is stretched more compared to other single strands, which is due to the lack of tendency of this single strand for internal hydrogen bonds and regular spiral structure resulting from a strong internal π-π stacking interaction. As a result, single strand of adenine is more accessible to detect target single strands for use in microarrays.

The effect of GGC and CAG repeat polymorphisms on the androgen receptor gene in response to finasteride therapy in men with androgenetic alopecia.

Background:It should be assessed whether the polymorphisms on androgen receptor gene can affect therapeutic response to androgenetic alopecia (AGA) medications. We aimed to find a link between polymorphisms on the androgen receptor gene (including the number of triple sequences of cytosine, adenine, and guanine [CAG] and guanine-guanine-cytosine [GGC]) and response to treatment with finasteride in male patients.Materials and Methods:This case–control study was performed on 25 consecutive male patients with hereditary AGA and 25 sex-matched healthy individuals without AGA. The complete sequence of the gene was extracted from the NCBI database. To replicate the samples, real-time polymerase chain reaction technique was used for the pointed gene and the results were confirmed by the sequencing technique.Results:The mean number of CAG sequences in two groups with and without baldness, was 23.16 ± 0.47 and 23.04 ± 0.67. For GGC sequencing with and without baldness, mean count was 22.22 ± 1.45 and 19.92 ± 81.2, respectively, which was significantly higher in the group with baldness. There was no association between number of CAG sequence and improvement in hair loss or the level of patients' satisfaction, but lower number of GGC sequences was associated with higher rate of stopping hair loss, more new hair growth, higher level of satisfaction, and more clinical response to finasteride and clinical improvement in AGA patients.Conclusion:Counting of GGC sequence in the gene encoding the androgen receptor is associated with an increase in odds of baldness and a decrease in the response rate to finasteride in AGA patients.

Detection of p53 Gene Mutation (Single-Base Mismatch) Using a Fluorescent Silver Nanoclusters.

P53 mutation was detected through the application of a biosensing approach based on the decrease in the fluorescence of oligonucleotide-templated silver nanoclusters (DNA-AgNCs). To this end specific DNA scaffolds of two various nucleotide fragments were used. One of the scaffolds was enriched with two cytosine sequence fragment (C12). This led to DNA-AgNCs with a fluorescence intensity through chemical reduction, while the other scaffold acted as the probe fragment (5- GTAGATGGCCATGGCGCGGACGCGGGTG-3). This latter scaffold selectively bound to the specific p53 site. Thus, resulting AgNCs demonstrated decreased fluorescence upon binding to single-base mismatching targets, and this behavior was found to be linearly proportional to the concentration of mutated p53 from 5 to 350nM and the approach was found to be able to detect concentrations as low as 1.3nM.

Effects of 5-Hydroxymethylcytosine Epigenetic Modifications within the VEGF Promoter Region on G-Quadruplex and I-Motif DNA Structure and Stability

Epigenetic modifications to DNA base sequences may regulate gene expression. CpG islands can contain methylated (5mC) or hydroxymethylated (5hmC) cytosine. Most CpG islands are found primarily in promoter regions that may also contain a high number of repeated cytosines and/or guanines. G-quadraplexes (G4) and i-motifs (iM) are two unique DNA secondary structures that can form in repeating sequences of either guanine or cytosine, respectively. Both G4 and iM sequences may contain CpG sequences that can be methylated or hydroxymethylated. The effects of CpG islands on DNA secondary structures were determined by incorporating a single 5hmC at varying positions in the Vascular Endothelial Growth Factor (VEGF) G4 and iM sequences. An Olis DSM-20 spectropolarimeter and a Cary 100 UV-visible spectrometer were used to monitor the effect of 5hmC on G4 and iM thermal stability. Two of the three 5hmC-containing loops showed a notable decrease in stability for G4's and increased intermolecular structure formation. Contrastingly, the iM stability increased when 5hmC was incorporated into its sequence. Additionally, there was little change in the iM pka. In summary, our results suggest the 5hmC has little effect on iM structures, but can destabilize the G4's.

Repair of a Dimeric Azetidine Related to the Thymine–Cytosine (6‐4) Photoproduct by Electron Transfer Photoreduction

AbstractPhotolyases are intriguing enzymes that take advantage of sunlight to restore lesions like cyclobutane pyrimidine dimers or (6‐4) photoproducts. This work focused on the photoreductive process responsible for splitting of the azetidine ring proposed to occur during (6‐4) photoproduct repair at a thymine–cytosine sequence. A model compound formed by photocycloaddition between thymine and 6‐azauracil has been designed to mimic the elusive azetidine intermediate. The photoinduced electron transfer process has been investigated by means of steady‐state and time‐resolved fluorescence using photosensitizers with oxidation potentials in the singlet excited state ranging from −3.3 to −2.1 V vs. SCE. Azetidine ring splitting and recovery of “repaired” bases were proven by HPLC analysis.

A fluorescent aptasensor for sensitive analysis oxytetracycline based on silver nanoclusters.

A fluorescent aptasensor for detection of oxytetracycline (OTC) was presented based on fluorescence quenching of DNA aptamer-templated silver nanoclusters (AgNCs). The specific DNA scaffolds with two different nucleotides fragments were used: one was enriched with a cytosine sequence fragment (C12) that could produce DNA-AgNCs via a chemical reduction method, and another was the OTC aptamer fragment that could selectively bind to the OTC antibiotic. Thus, the as-prepared AgNCs could exhibit quenched fluorescence after binding to the target OTC. The fluorescence ratio of the DNA-AgNCs was quenched in a linearly proportional manner to the concentration of the target in the range of 0.5 nM to 100 nM with a detection limit of 0.1 nM. This proposed nanobiosensor was demonstrated to be sensitive, selective, and simple, introducing a viable alternative for rapid determination of toxin OTC in honey and water samples. Copyright © 2016 John Wiley & Sons, Ltd.

A Novel Label-Free microRNA-155 Detection on the Basis of Fluorescent Silver Nanoclusters.

In this paper, a new approach for microRNA-155 (miRNA-155) detection was described based on the fluorescence quenching of oligonucleotide-templated silver nanoclusters (DNA-AgNCs). The specific DNA scaffold with two different nucleotides fragments were used: one was enriched with a cytosine sequence fragment (C12) that could result in DNA-AgNCs with a high quantum yield via a chemical reduction method, and the other was the probe fragment (5- CUGUUAAUGCUAAUCGUG-3) which could selectively bind to the miRNA-155. Thus, the as-prepared AgNCs could exhibit quenched fluorescence when binding to the target miRNA-155. The fluorescence ratio of the DNA-AgNCs was quenched in a linearly proportional manner to the concentration of the target in the range of 0.2 nM to 30 nM with a detection limit of 0.1 nM.

DIFFERENTIAL METHYLATION METHODS IN MULTI-CONTEXT ORGANISMS

DNA methylation is an epigenetic modification that has the ability to alter gene expression without any change in the DNA sequence. DNA methylation occurs when a methyl chemical group attaches to cytosine bases on the DNA sequence. In mammals, DNA methylation primarily occurs at CG sites, when a cytosine is followed by a guanine in the DNA sequence. In plants, DNA methylation can also occur in other cytosine sequences, such as when a cytosine is not followed directly by a guanine. Many of the statistical methods that have been developed to estimate methylation levels and test differential methylation in whole-genome bisulfite sequencing studies incorporate the observed correlation between methylation levels of neighboring cytosine sites. However, most of these methods have been applied to human studies, where only CG sites are investigated. In this study, we focus on plant studies and show that the correlation between methylation levels at neighboring sites depends on the DNA sequence immediately following the cytosine. We investigate the importance of accounting for these differences in the correlation structure by comparing the performance of three existing methods (MethylSig, MAGI, and M3D) in plants.

Effective detection and cell imaging of prion protein with new prepared targetable yellow-emission silver nanoclusters

Silver nanoclusters (AgNCs) have gained considerable attention over a wide range from chemical detection to bioimaging applications. Herein we developed a facile way to synthesize water-soluble, intense fluorescent targetable AgNCs with yellow-emission by using DNA via a chemical reduction method. The sequence of DNA was designed to contain two fragments, one of which is the DNA aptamer fragment which can selectively bind to the target, and the other is rich in cytosine sequence fragments that can result in a high quantum efficiency. The as-prepared targetable AgNCs (Apt-AgNCs) through the reduction of silver ions (Ag(+)) by NaBH(4) have a very strong fluorescence emission, which, was greatly quenched upon specific binding to prion protein (PrP(C)). As a result, Apt-AgNCs were demonstrated as a novel, effective and sensitive probe for both detection and intracellular fluorescent imaging with high selectivity through the strong binding affinity between the aptamer and its target.

Label-free DNA detection on the basis of fluorescence resonance energy transfer from oligonucleotide-templated silver nanoclusters to multi-walled carbon nanotubes

In this paper, a new approach for respiratory syncytial virus (RSV) gene sequence detection was described based on the fluorescence resonance energy transfer (FRET) from oligonucleotide-templated silver nanoclusters (DNA–AgNCs) to multi-walled carbon nanotubes (MWCNTs). The specific DNA scaffold combines two fragments: one is enriched with a cytosine sequence fragment (C12) that can result in DNA–AgNCs with a high quantum yield via a chemical reduction method, and the other is the probe fragment (5′-AAA AAT GGG GCA AAT A-3′) which can selectively bind to the gene for RSV. Thus, the as-prepared AgNCs can exhibit enhanced fluorescence when binding to the target DNA sequence and forming a double helix. Because of the introduction of MWCNTs, which can quench the fluorescence of the DNA–AgNCs with an extraordinarily high quenching efficiency (85.8%), a relatively high signal-to-background ratio was achieved. The fluorescence ratio of the DNA–AgNCs was enhanced in a linearly proportional manner to the concentration of the target in the range of 31.25 nM to 2.00 μM with a detection limit (3σ) of 24.00 nM.

Quinacrine and 9-Amino Acridine Inhibit B-Z and B-HL Form DNA Conformational Transitions

The interaction of quinacrine and 9-amino acridine with right-handed B-form, left-handed Z-form, and left-handed protonated (H(L))-form structures of polydG-me(5)dC was investigated by circular dichroism and absorption spectral analysis. Both the compounds bind strongly to the B-form structure and convert the Z-form and H(L)-form back to the bound right-handed form. Circular dichroic data revealed that the conformation at the binding site is right-handed even though adjacent regions of the polynucleotide may have left-handed conformation. The rate and extent of B-form-to-Z-form transition were decreased in the presence of these compounds. Scatchard analysis revealed that both quinacrine and 9-amino acridine bind strongly to the polynucleotide in the B-form in a noncooperative manner, in sharp contrast to the highly cooperative binding to the Z-form and H(L)-form. Results indicated that the cooperative binding of these drugs with the Z-form and the H(L)-forms was associated with a sequential conversion of the polynucleotide from a left-handed to a bound right-handed conformation. Experimental data enabled the calculation of the number of base pairs of Z-form (7-8 with quinacrine and 9-amino acridine) and H(L)-form (4 and 25, respectively, with quinacrine and 9-amino acridine) that adopt a right-handed conformation for each bound ligand. As these compounds are known to bind preferentially to alternating guanine--cytosine sequences, which are capable of easily undergoing the B-to-Z or B-to-H(L) transition, these effects may be important in understanding their biological activities.