QuadBase: genome-wide database of G4 DNA—occurrence and conservation in human, chimpanzee, mouse and rat promoters and 146 microbes

Emerging evidence indicates the importance of G-quadruplex motifs as drug targets. [Stuart A. Borman, Ascent of quadruplexes—nucleic acid structures become promising drug targets. Chem. Eng. News, 2007;85, 12–17], which stems from the fact that these motifs are present in a surprising number of promoters wherein their role in controlling gene expression has been demonstrated for a few. We present a compendium of quadruplex motifs, with particular focus on their occurrence and conservation in promoters—QuadBase. It is composed of two parts (EuQuad and ProQuad). EuQuad gives information on quadruplex motifs present within 10 kb of transcription starts sites in 99 980 human, chimpanzee, rat and mouse genes. ProQuad contains quadruplex information of 146 prokaryotes. Apart from gene-specific searches for quadruplex motifs, QuadBase has a number of other modules. ‘Orthologs Analysis’ queries for conserved motifs across species based on a selected reference organism; ‘Pattern Search’ can be used to fetch specific motifs of interest from a selected organism using user-defined criteria for quadruplex motifs, i.e. stem, loop size, etc. ‘Pattern Finder’ tool can search for motifs in any given sequence. QuadBase is freely available to users from non-profit organization at http://quadbase.igib.res.in/

Quadruplex DNA: sequence, topology and structure.

G-quadruplexes are higher-order DNA and RNA structures formed from G-rich sequences that are built around tetrads of hydrogen-bonded guanine bases. Potential quadruplex sequences have been identified in G-rich eukaryotic telomeres, and more recently in non-telomeric genomic DNA, e.g. in nuclease-hypersensitive promoter regions. The natural role and biological validation of these structures is starting to be explored, and there is particular interest in them as targets for therapeutic intervention. This survey focuses on the folding and structural features on quadruplexes formed from telomeric and non-telomeric DNA sequences, and examines fundamental aspects of topology and the emerging relationships with sequence. Emphasis is placed on information from the high-resolution methods of X-ray crystallography and NMR, and their scope and current limitations are discussed. Such information, together with biological insights, will be important for the discovery of drugs targeting quadruplexes from particular genes

Putative DNA G-quadruplex formation within the promoters of Plasmodium falciparum var genes

Background. Guanine-rich nucleic acid sequences are capable of folding into an intramolecular four-stranded structure called a G-quadruplex. When found in gene promoter regions, G-quadruplexes can downregulate gene expression, possibly by blocking the transcriptional machinery. Here we have used a genome-wide bioinformatic approach to identify Putative G-Quadruplex Sequences (PQS) in the Plasmodium falciparum genome, along with biophysical techniques to examine the physiological stability of P. falciparum PQS in vitro. Results. We identified 63 PQS in the non-telomeric regions of the P. falciparum clone 3D7. Interestingly, 16 of these PQS occurred in the upstream region of a subset of the P. falciparum var genes (group B var genes). The var gene family encodes PfEMP1, the parasite’s major variant antigen and adhesin expressed at the surface of infected erythrocytes, that plays a key role in malaria pathogenesis and immune evasion. The ability of the PQS found in the upstream regions of group B var genes (UpsB-Q) to form stable Gquadruplex structures in vitro was confirmed using 1H NMR, circular dichroism, UV spectroscopy, and thermal denaturation experiments. Moreover, the synthetic compound BOQ1 that shows a higher affinity for DNA forming quadruplex rather than duplex structures was found to bind with high affinity to the UpsB-Q. Conclusions. This is the first demonstration of non-telomeric PQS in the genome of P. falciparum that form stable G-quadruplexes under physiological conditions in vitro. These results allow the generation of a novel hypothesis that the G-quadruplex sequences in the upstream regions of var genes have the potential to play a role in the transcriptional control of this major virulence-associated multi-gene family

The HSV-1 ICP27 RGG box specifically binds flexible, GC-rich sequences but not G-quartet structures

Herpes simplex virus 1 (HSV-1) protein ICP27, an important regulator for viral gene expression, directly recognizes and exports viral RNA through an N-terminal RGG box RNA binding motif, which is necessary and sufficient for RNA binding. An ICP27 N-terminal peptide, including the RGG box RNA binding motif, was expressed and its binding specificity was analyzed using EMSA and SELEX. DNA oligonucleotides corresponding to HSV-1 glycoprotein C (gC) mRNA, identified in a yeast three-hybrid analysis, were screened for binding to the ICP27 N-terminal peptide in EMSA experiments. The ICP27 N-terminus was able to bind most gC substrates. Notably, the ICP27 RGG box was unable to bind G-quartet structures recognized by the RGG domains of other proteins. SELEX analysis identified GC-rich RNA sequences as a common feature of recognition. NMR analysis of SELEX and gC sequences revealed that sequences able to bind to ICP27 did not form secondary structures and conversely, sequences that were not able to bind to ICP27 gave spectra consistent with base-pairing. Therefore, the ICP27 RGG box is unique in its recognition of nucleic acid sequences compared to other RGG box proteins; it prefers flexible, GC-rich substrates that do not form stable secondary structures

Remarkable stability of an instability-prone lentiviral vector plasmid in Escherichia coli Stbl3

Large-scale production of plasmid DNA to prepare therapeutic gene vectors or DNA-based vaccines requires a suitable bacterial host, which can stably maintain the plasmid DNA during industrial cultivation. Plasmid loss during bacterial cell divisions and structural changes in the plasmid DNA can dramatically reduce the yield of the desired recombinant plasmid DNA. While generating an HIV-based gene vector containing a bicistronic expression cassette 5′-Olig2cDNA-IRES-dsRed2-3′, we encountered plasmid DNA instability, which occurred in homologous recombination deficient recA1 Escherichia coli strain Stbl2 specifically during large-scale bacterial cultivation. Unexpectedly, the new recombinant plasmid was structurally changed or completely lost in 0.5 L liquid cultures but not in the preceding 5 mL cultures. Neither the employment of an array of alternative recA1 E. coli plasmid hosts, nor the lowering of the culture incubation temperature prevented the instability. However, after the introduction of this instability-prone plasmid into the recA13E. coli strain Stbl3, the transformed bacteria grew without being overrun by plasmid-free cells, reduction in the plasmid DNA yield or structural changes in plasmid DNA. Thus, E. coli strain Stbl3 conferred structural and maintenance stability to the otherwise instability-prone lentivirus-based recombinant plasmid, suggesting that this strain can be used for the faithful maintenance of similar stability-compromised plasmids in large-scale bacterial cultivations. In contrast to Stbl2, which is derived wholly from the wild type isolate E. coli K12, E. coli Stbl3 is a hybrid strain of mixed E. coli K12 and E. coli B parentage. Therefore, we speculate that genetic determinants for the benevolent properties of E. coli Stbl3 for safe plasmid propagation originate from its E. coli B ancestor

Ionic modulation of QPX stability as a nano - switch regulating gene expression in neurons

G-quadruplexes (G-QPX) have been the subject of intense research due to their unique structural configuration and potential applications, particularly their functionality in biological process as a novel type of nano–switch. They have been found in critical regions of the human genome such as telomeres, promoter regions, and untranslated regions of RNA. About 50% of human DNA in promoters has G-rich regions with the potential to form G-QPX structures. A G-QPX might act mechanistically as an ON/OFF switch, regulating gene expression, meaning that the formation of G-QPX in a single strand of DNA disrupts double stranded DNA, prevents the binding of transcription factors (TF) to their recognition sites, resulting in gene down-regulation. Although there are numerous studies on biological roles of G-QPXs in oncogenes, their potential formation in neuronal cells, in particular upstream of transcription start sites, is poorly investigated. The main focus of this research is to identify stable G-QPXs in the 97bp active promoter region of the choline acetyltransferase (ChAT) gene, the terminal enzyme involved in synthesis of the neurotransmitter acetylcholine, and to clarify ionic modulation of G-QPX nanostructures through the mechanism of neural action potentials. Different bioinformatics analyses (in silico), including the QGRS, quadparser and G4-Calculator programs, have been used to predict stable G-QPX in the active promoter region of the human ChAT gene, located 1000bp upstream from the TATA box. The results of computational studies (using those three different algorithms) led to the identification of three consecutive intramolecular G-QPX structures in the negative strand (ChAT G17-2, ChAT G17, and ChAT G29) and one intramolecular G-QPX structure in the positive strand (ChAT G30). Also, the results suggest the possibility that nearby G-runs in opposed DNA strands with a short distance of each other may be able to form a stable intermolecular G-QPX involving two DNA complementary strands (ds ChAT G21). Formation of G-QPX structures, by blocking the availability of the transcription factor binding site (TFBS) on double stranded DNA, can interfere with transcriptional activation. This suggests that there is competition between TFBS binding to dsDNA and the conversion to high order non-B form secondary structures (G-QPXs) in the active promoter region. TFBS mapping analysis of the active promoter region of the human ChAT gene revealed that it contains multiple consensus AP-2a and Sp1 binding sites and consensus sites for other TF, including multiple sites for GR-alpha, Pax-5, p53 and GC box proteins. To get a better understanding of how modulation of G-QPX structures might affect the ChAT promoter activity, an artificial GFP reporter vector (modified GFP) was constructed, synthesized and used for reporter gene measurement. As known human ChAT promoter activators, nerve growth factors (HNGF and TGB) and cytokines (IL-ß and TNF-a) were used for activation of the artificial promoter driving GFP. Also, the G-QPX stabilizing drug TMPYP4 and aconitine, a Na+ channel opening drug, were used as G-QPX stability modulating factors. It was observed that aconitine potentiated the action of the transcriptional activator NGF, suggesting that the effect of sodium is contrary to that of TMPYP4, i.e., that an increase in promoter activity may be due to instability of G-QPX structures in a high Na+ environment, which results in melting these structures, enabling dsDNA formation required for the binding of TF to their recognition sites for initiation of transcription. The results were confirmed in several independent sets of experiments, using GFP reporter gene measurement by plate reader, by flow cytometry and using fluorescent microscopy. Moreover, quantitative RT-PCR was conducted to evaluate the effect of the same factors under similar conditions on the actual ChAT mRNA expression. It was observed that TMPY4 knocked down the ChAT mRNA expression by 87%, suggesting that G-QPX stabilization inhibits promoter activity as expected and that aconitine along with HNGF increases ChAT mRNA expression up to 2.8 fold. Aconitine-mediated influx of Na+ ions, possibly by inhibiting the formation of stable G-QPX structures, resulted in an Unique G-QPX structures can be stabilized with certain metal cations or small cationic molecule ligands such as TMPYP4, through occupying the space between the layers of G-tetrads. Although G-QPX are reported to have high stability in potassium solution, the diversity of G-QPX structures (due to diversity in sequence and size of G-runs, sequence and size of loops) will lead to diversity in physical behavior of G-QPX structures. Therefore, to get a clear image of folding topology and stability of identified G-QPX structures, physical studies including CD spectroscopy and AFM imaging were conducted. CD results showed that the identified ChAT G-QPX structures formed a hybrid, stable configuration in potassium environment (10mM) while being instable in sodium solution (100mM). AFM imaging demonstrated star-shaped structures (involving clusters of DNA strands) due to incubation with TMPYP4, where a greater number of these G-rich sequences have converted to G-QPX structures. The results of both an artificial engineered reporter gene system and actual ChAT mRNA expression (in vitro), plus physical characterization studies, strongly support the novel hypothesis that a neural action potential ionic mechanism regulates G-QPX formation/ deformation in the promoter region, due to movement of monovalent cations across the membrane, which is consistent with gene silencing and expression during neuronal resting and firing

Biosynthesis of a G-Quadruplex—forming sequence and its stabilization by ligands

In addition to the Watson and Crick B-form duplex DNA, G-quadruplexes are four-stranded DNA structures formed in vivo by the self-assembly of guanine-rich sequences. These can be formed by one, two or four separate strands of DNA and present a diversity of topologies, defined by the strand orientation, loop size and sequence. G-quadruplexes can be found in telomeres, immunoglobulin switch regions and gene promoter regions. The biological relevant location on the genome makes these high-order structures an attractive target for drug design and the development of highly specific ligands that bind and stabilize G-quadruplex with therapeutic activity. Herein, the biosynthesis of a novel G-rich quadruplex-forming DNA sequence 58Sγ3 is described by plasmid amplification. The recovery and purification of 58Sγ3 oligonucleotide using size-exclusion chromatography is presented. The G-quadruplex formation is promoted and its topology is determined by circular dichroism. The stabilization of the G-quadruplex structure with quinoline and naphthalene-based derivatives is studied using melting analysis, G4-FID and PCR-stop assays. The results suggest that 58Sγ3 folds into a parallel-stranded G-quadruplex structure in 500 mM KCl buffer and that naphthalene-based ligands bind and stabilize the G-quadruplex structure. The ligands are also found to be quadruplex-specific over duplex DNA and inhibit Taq DNA polymerase. This work provides evidence for G-quadruplex formation within the immunoglobulin switch regions. Furthermore, it is suggested that the novel ligands here reported act as potent specific G-quadruplex binders and may also potentially be used to inhibit genes transcription in tumor cells.Além da forma B Watson e Crick do ADN duplex, os G-quadruplexes são estruturas de ADN de quatro cadeias, formadas in vivo pela auto-associação de sequências ricas em guaninas. Estas podem ser formadas por uma, duas ou quatro cadeias distintas de ADN e apresentar uma diversidade de topologias, definidas pela orientação da cadeia, tamanho dos loops e a sequência. G-quadruplexes podem ser encontrados nos telómeros, regiões de troca das imunoglobulinas e nas regiões dos promotores génicos. A localização biologicamente relevante no genoma faz com que estas estruturas altamente ordenadas sejam um alvo atrativo do desenho de fármacos e o desenvolvimento de ligandos altamente específicos que ligam e estabilizam o G-quadruplex com ação terapêutica. Neste trabalho, descreve-se a biossíntese da nova sequência de ADN rica em guaninas e formadora de G-quadruplex 58Sγ3, utilizando amplificação por plasmídeo. A recuperação e purificação do oligonucleótido 58Sγ3 é efetuada por cromatografia de exclusão molecular. A formação de G-quadruplex é promovida e a sua topologia é determinada por dicroísmo circular. A estabilização da estrutura do G-quadruplex com ligandos derivados de quinolina e naftaleno é estudada utilizando ensaios de estabilização térmica no dicroísmo circular, G4-FID e PCR-stop. Os resultados sugerem que 58Sγ3 adota uma estrutura G-quadruplex paralela em tampão 500 mM KCl e que os ligandos de naftaleno ligam e estabilizam a estrutura do G-quadruplex. Os ligandos demonstraram também ser específicos do G-quadruplex em relação ao ADN duplex além de inibir a Taq ADN polimerase. Este trabalho fornece evidência da formação de G-quadruplex nas regiões de troca das imunoglobulinas. Além disso, sugere que os derivados de naftaleno atuam como ligandos do G-quadruplex e que podem ser potencialmente utilizados para inibir a transcrição de genes em células tumorais

Inhibition of human telomerase by a G-quadruplex-interaction compound

Certain non-nucleoside compounds that will selectively inhibit telomerase by targeting the nucleic add structures, such as G-quadruplexes, that may be associated with human telomeres or telomerase have been identified. Inhibition of human telomerase by two perylenetetracarboxylic acid diimides and a carbocyanine has been demonstrated. 1H-NMR studies have evidenced the stabilization of a G-quadruplex by the perylenetetracarboxylic acid diimide compounds and provided evidence that these and structurally related compounds inhibit the telomerase enzyme by a mechanism consistent with interaction with G-quadruplex structures.Board of Regents, University of Texas Syste