Distinctive aspects of ligand binding to G4 DNA structure flanked by the double helix.

Except for telomeres, G4 DNA structures in the human genome can be formed only within the context of double-stranded DNA. DNA duplexes flanking the G4 structure may potentially affect the G4 architecture and the binding of G4-specific ligands. Here, we examine the interaction of TMPyP4, NMM, and PDS ligands with three structures formed by the same DNA fragment containing the (GGGT) sequence: the G4 in duplex (dsG4), G4 in single-stranded DNA (ssG4) and perfect duplex DNA (ds).

Quenching of G4-DNA intrinsic fluorescence by ligands.

G-quadruplex (G4) structures formed by the guanine-rich DNA regions exhibit several distinctive optical properties, including UV absorption and circular dichroism spectra. Some G4 DNA possess intrinsic UV fluorescence whose origin is not completely clear to date. In this work, we study the effect of TMPyP4 and Methylene Blue on the intrinsic fluorescence of the dimeric G4 DNA structure formed by two d(GT) sequences.

Taq-Polymerase Stop Assay to Determine Target Selectivity of G4 Ligands in Native Promoter Sequences of , , and Oncogenes.

Computational and high-throughput experimental methods predict thousands of potential quadruplex sequences (PQSs) in the human genome. Often these PQSs contain more than four G-runs, which introduce additional uncertainty into the conformational polymorphism of the G4 DNA. G4-specific ligands, which are currently being actively developed as potential anticancer agents or tools for studying G4 structures in genomes, may preferentially bind to specific G4 structures over the others that can be potentially formed in the extended G-rich genomic region.

Differential Impact of Random GC Tetrad Binding and Chromatin Events on Transcriptional Inhibition by Olivomycin A.

Olivomycin A (OA), an antibiotic of the aureolic acid family, interferes with gene transcription upon forming complexes with GC-rich regions in the DNA minor groove. We demonstrate that the mechanism of transcriptional deregulation is not limited to OA interaction with GC-containing binding sites for transcription factors. Using electrophoretic mobility shift assays and DNAse I footprinting of cytomegalovirus (CMV) promoter fragments carrying OA-preferred GC tetrads (CMVwt), we showed OA binding specifically to GC islands.

Genome Assembly and Sex-Determining Region of Male and Female × .

The genus is presented by dioecious species, and it became a promising object to study the genetics of sex in plants. In this work, genomes of male and female × individuals were sequenced for the first time. To achieve high-quality genome assemblies, we used Oxford Nanopore Technologies and Illumina platforms.

Genome and Transcriptome Sequencing of × Identified Sex-Associated Allele-Specific Expression of the Gene.

Transcriptome sequencing of leaves, catkin axes, and flowers from male and female trees of × and genome sequencing of the same plants were performed for the first time. The availability of both genome and transcriptome sequencing data enabled the identification of allele-specific expression. Such an analysis was performed for genes from the sex-determining region (SDR).

Oxidative probing of the G4 DNA structure induced in double-stranded DNA by molecular crowding or pyridostatin.

Major advances have been made recently in the application of the highly selective G4 DNA ligand pyridostatin (PDS) for targeting and visualization of this noncanonical DNA structure in eukaryotic genomes. However, the interaction of PDS with the G4 structure constrained by double-stranded DNA has not yet been analyzed. Here, we induced folding of G4 structures in double-stranded DNA promoter fragments of several oncogenes by annealing the DNA under molecular crowding conditions created by polyethylene glycol (PEG) or in the presence of PDS.

Recurrent Potential G-Quadruplex Sequences in Archaeal Genomes.

Evolutionary conservation or over-representation of the potential G-quadruplex sequences (PQS) in genomes are usually considered as a sign of the functional relevance of these sequences. However, uneven base distribution (GC-content) along the genome may along the genome may result in seeming abundance of PQSs over average in the genome. Apart from this, a number of other conserved functional signals that are encoded in the GC-rich genomic regions may inadvertently result in emergence of G-quadruplex compatible sequences.

Discrimination between G/C Binding Sites by Olivomycin A Is Determined by Kinetics of the Drug-DNA Interaction.

Olivomycin A (OA) exerts its cytotoxic potency due to binding to the minor groove of the G/C-rich DNA and interfering with replication and transcription. Screening of the complete set of tetranucleotide G/C sites by electrophoretic mobility gel shift assay (EMSA) revealed that the sites containing central GC or GG dinucleotides were able to bind OA, whereas the sites with the central CG dinucleotide were not. However, studies of equilibrium OA binding in solution by fluorescence, circular dichroism and isothermal titration calorimetry failed to confirm the sequence preference of OA, indicating instead a similar type of complex and comparable affinity of OA to all G/C binding sites.

Tumor suppressor properties of the small C-terminal domain phosphatases in non-small cell lung cancer.

Non-Small Cell Lung Cancer (NSCLC) is responsible for the majority of deaths caused by cancer. Small C-terminal domain (CTD) phosphatases (SCP), CTDSP1, CTDSP2 and CTDSPL (CTDSPs) belong to SCP/CTDSP subfamily and are involved in many vital cellular processes and tumorigenesis. High similarity of their structures suggests similar functions.

Pan-Cancer Analysis of TCGA Data Revealed Promising Reference Genes for qPCR Normalization.

Quantitative PCR (qPCR) remains the most widely used technique for gene expression evaluation. Obtaining reliable data using this method requires reference genes (RGs) with stable mRNA level under experimental conditions. This issue is especially crucial in cancer studies because each tumor has a unique molecular portrait.

Conformational features of intramolecular G4-DNA constrained by single-nucleotide loops.

Conformation of the telomeric DNA fragment dG(TTAG) depends on multiple factors including solution conditions, length, and the nucleotide sequence of the flanking regions. In potassium solution, this sequence tends to adopt hybrid (3 + 1) G-quadruplex (G4) Form 1 or Form 2 conformation contingent on the flanking nucleotides. Theoretically, other (3 + 1) G4 folds (beyond Forms 1 and 2) are not sterically forbidden, but are presumably energetically disfavored.

Deep Sequencing Revealed a CpG Methylation Pattern Associated With Suppression in Breast Cancer.

Hypermethylation of promoter CpG islands is generally recognized epigenetic mechanism responsible for gene silencing in cancer. However, molecular details on how this epigenetic mark triggers the process of gene downregulation are still elusive. Here, we used deep bisulfite sequencing and qPCR analysis to investigate the pattern of CpG methylation of promoter region and its association with the gene expression level in 16 paired breast cancer (BC) samples of different clinical stages.

Divalent cations are dispensable for binding to DNA of a novel positively charged olivomycin A derivative.

The current model of binding of the antitumor antibiotic olivomycin A (1) to GC-rich DNA regions presumes that coordination of the magnesium divalent cation with drug dimers is necessary for binding of 1 into the minor groove of the DNA duplex. Previously we have synthesized the derivatives of 1 termed 'short acid' (2) and its N,N-dimethylaminoethylamide (3). The latter compound demonstrated an improved tolerance in vivo compared to 1 and good therapeutic potency in animal models.

Light-induced oxidation of the telomeric G4 DNA in complex with Zn(II) tetracarboxymethyl porphyrin.

Structure-specific ligands are convenient tools for the recognition, targeting or probing of non-canonical DNA structures. Porphyrin derivatives exhibit a preference for interaction with G-quadruplex (G4) structures over canonical duplex DNA and are able to cause photoinducible damage to nucleic acids. Here, we show that Zn(II) 5,10,15,20-tetrakis(N-carboxymethyl-4-pyridinium)porphyrin ( ZNP1: ) interacts with different conformations of the telomeric sequence d(TAGGG(TTAGGG)) at submicromolar concentrations without any detectible disturbance of the particular fold.

Identification of Novel Epigenetic Markers of Prostate Cancer by NotI-Microarray Analysis.

A significant need for reliable and accurate cancer diagnostics and prognosis compels the search for novel biomarkers that would be able to discriminate between indolent and aggressive tumors at the early stages of disease. The aim of this work was identification of potential diagnostic biomarkers for characterization of different types of prostate tumors. NotI-microarrays with 180 clones associated with chromosome 3 genes/loci were applied to determine genetic and epigenetic alterations in 33 prostate tumors.

SECIS-binding protein 2, a key player in selenoprotein synthesis, is an intrinsically disordered protein.

Selenocysteine (Sec) is co-translationally incorporated into selenoproteins at a reprogrammed UGA codon. In mammals, this requires a dedicated machinery comprising a stem-loop structure in the 3' UTR RNA (the SECIS element) and the specific SECIS Binding Protein 2. In this report, disorder-prediction methods and several biophysical techniques showed that ca.

Distinctive structures between chimpanzee and human in a brain noncoding RNA.

Human accelerated region 1 (HAR1) is a short DNA region identified recently to have evolved the most rapidly among highly constrained regions since the divergence from our common ancestor with chimpanzee. It is transcribed as part of a noncoding RNA specifically expressed in the developing human neocortex. Employing a panoply of enzymatic and chemical probes, our analysis of HAR1 RNA proposed a secondary structure model differing from that published.

2-aminopurine fluorescence: discrimination between specific and unspecific ligand binding to the kissing-loop dimer of the HIV-1 RNA.

The fluorescent 2-aminopurine probe (2-AP) incorporated into the loop of 23-mer RNA hairpin of HIV-1 genome dimerization initiation site (DIS) was used for discrimination of specific and unspecific binding of paromomycin and spermine to the kissing loop dimer (KD) formed in solution. While both ligands stabilized the KD RNA structure, only paromomycin binding resulted in significant increase of 2-AP fluorescence. These observations suggest that the 2-AP fluorescent RNA construct might be useful for selecting ligands specifically binding the HIV-1 kissing loop RNA dimer.

A protein component at the heart of an RNA machine: the importance of protein l27 for the function of the bacterial ribosome.

Deletion of the gene for protein L27 from the E. coli chromosome results in severe defects in cell growth. This deficiency is corrected by the expression of wild-type (wt) protein L27 from a plasmid.