664 research outputs found

    Predicting and understanding the stability of G-quadruplexes

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    Motivation: G-quadruplexes are stable four-stranded guanine-rich structures that can form in DNA and RNA. They are an important component of human telomeres and play a role in the regulation of transcription and translation. The biological significance of a G-quadruplex is crucially linked with its thermodynamic stability. Hence the prediction of G-quadruplex stability is of vital interest

    High-Resolution Three-Dimensional NMR Structure Of The KRAS Proto-Oncogene Promoter Reveals Key Features Of A G-Quadruplex Involved In Transcriptional Regulation

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    Non-canonical base pairing within guanine-rich DNA and RNA sequences can produce G-quartets, whose stacking leads to the formation of a G-quadruplex (G4). G4s can coexist with canonical duplex DNA in the human genome and have been suggested to suppress gene transcription, and much attention has therefore focused on studying G4s in promotor regions of disease-related genes. For example, the human KRAS proto-oncogene contains a nuclease-hypersensitive element located upstream of the major transcription start site. The KRAS nuclease-hypersensitive element (NHE) region contains a G-rich element (22RT; 5′-AGGGCGGTGTGGGAATAGGGAA-3′) and encompasses a Myc-associated zinc finger-binding site that regulates KRAS transcription. The NEH region therefore has been proposed as a target for new drugs that control KRAS transcription, which requires detailed knowledge of the NHE structure. In this study, we report a high-resolution NMR structure of the G-rich element within the KRAS NHE. We found that the G-rich element forms a parallel structure with three G-quartets connected by a four-nucleotide loop and two short one-nucleotide double-chain reversal loops. In addition, a thymine bulge is found between G8 and G9. The loops of different lengths and the presence of a bulge between the G-quartets are structural elements that potentially can be targeted by small chemical ligands that would further stabilize the structure and interfere or block transcriptional regulators such as Myc-associated zinc finger from accessing their binding sites on the KRAS promoter. In conclusion, our work suggests a possible new route for the development of anticancer agents that could suppress KRAS expression

    Interactions Between Spermine-Derivatized Tentacle Porphyrins And The Human Telomeric DNA G-Quadruplex

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    G-rich DNA sequences have the potential to fold into non-canonical G-Quadruplex (GQ) structures implicated in aging and human diseases, notably cancers. Because stabilization of GQs at telomeres and oncogene promoters may prevent cancer, there is an interest in developing small molecules that selectively target GQs. Herein, we investigate the interactions of meso-tetrakis-(4-carboxysperminephenyl)porphyrin (TCPPSpm4) and its Zn(II) derivative (ZnTCPPSpm4) with human telomeric DNA (Tel22) via UV-Vis, circular dichroism (CD), and fluorescence spectroscopies, resonance light scattering (RLS), and fluorescence resonance energy transfer (FRET) assays. UV-Vis titrations reveal binding constants of 4.7 × 10⁶ and 1.4 × 10⁷ M⁻¹ and binding stoichiometry of 2–4:1 and 10–12:1 for TCPPSpm4 and ZnTCPPSpm4, respectively. High stoichiometry is supported by the Job plot data, CD titrations, and RLS data. FRET melting indicates that TCPPSpm4 stabilizes Tel22 by 36 ± 2 °C at 7.5 eq., and that ZnTCPPSpm4 stabilizes Tel22 by 33 ± 2 °C at ~20 eq.; at least 8 eq. of ZnTCPPSpm4 are required to achieve significant stabilization of Tel22, in agreement with its high binding stoichiometry. FRET competition studies show that both porphyrins are mildly selective for human telomeric GQ vs duplex DNA. Spectroscopic studies, combined, point to end-stacking and porphyrin self-association as major binding modes. This work advances our understanding of ligand interactions with GQ DNA

    Ab initio RNA folding

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    RNA molecules are essential cellular machines performing a wide variety of functions for which a specific three-dimensional structure is required. Over the last several years, experimental determination of RNA structures through X-ray crystallography and NMR seems to have reached a plateau in the number of structures resolved each year, but as more and more RNA sequences are being discovered, need for structure prediction tools to complement experimental data is strong. Theoretical approaches to RNA folding have been developed since the late nineties when the first algorithms for secondary structure prediction appeared. Over the last 10 years a number of prediction methods for 3D structures have been developed, first based on bioinformatics and data-mining, and more recently based on a coarse-grained physical representation of the systems. In this review we are going to present the challenges of RNA structure prediction and the main ideas behind bioinformatic approaches and physics-based approaches. We will focus on the description of the more recent physics-based phenomenological models and on how they are built to include the specificity of the interactions of RNA bases, whose role is critical in folding. Through examples from different models, we will point out the strengths of physics-based approaches, which are able not only to predict equilibrium structures, but also to investigate dynamical and thermodynamical behavior, and the open challenges to include more key interactions ruling RNA folding.Comment: 28 pages, 18 figure

    Analysis of G-Quadruplex Formation in mRNA Transcripts of Phospholemman/FXYD1

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    G-quadruplexes are higher-order nucleic acid structures formed by tetrads of guanine bases (G-tetrads) through non-canonical base interactions. Two G-tetrads are stabilised by a potassium-ion sandwiched between the tetrads. It has emerged from recent studies that G-quadruplexes occur widely throughout the human genome and have significant biological roles. In this study the FXYD1 pre-mRNA encoding the protein Phospholemman (PLM) is investigated. PLM is highly expressed in cardiomyocytes and forms a third subunit of the Na+/K+ pump (NKA). PLM is a major phosphorylation target and thus regulates NKA activity. FXYD1 pre-mRNA was investigated for its ability to form G-quadruplexes. By computational analysis, it was found that FXYD1 can fold into G-quadruplex and multiple sequence alignment of ortholog FXYD1 sequences indicated that G-quadruplex-forming potential is conserved in evolution, hinting at a potential regulatory mechanism of FXYD1 expression. Comparative analysis confirmed that FXYD1-009, a variant of FXYD1, is a product of alternative splicing of FXYD1’s pre-mRNA. G-quadruplex formation in human and bovine FXYD1-derived oligonucleotides was detected experimentally by non-denaturing poly acrylamide gel electrophoresis that showed an increased mobility rate of G-quadruplexes in contrast to controls. Further analysis by fluorescence emission spectroscopy confirmed G-quadruplex formation in the human and bovine FXYD1-oligonucleotides that was triggered by the presence of K+ ions. The results provided clear evidence of G-quadruplex formation in vitro and together with evolutionary conservation point to potential role in regulating expression of FXYD1 possibly through alternative splicing and thus regulate indirectly the activity of Na+/K+-ATPase. Further in-vivo works should address whether alternative splicing of FXYD1 to FXYD1-009 is associated with G-quadruplex formation

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

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    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

    Characterization of Aptamer-Protein Complexes by X-ray Crystallography and Alternative Approaches

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    Aptamers are oligonucleotide ligands, either RNA or ssDNA, selected for high-affinity binding to molecular targets, such as small organic molecules, proteins or whole microorganisms. While reports of new aptamers are numerous, characterization of their specific interaction is often restricted to the affinity of binding (KD). Over the years, crystal structures of aptamer-protein complexes have only scarcely become available. Here we describe some relevant technical issues about the process of crystallizing aptamer-protein complexes and highlight some biochemical details on the molecular basis of selected aptamer-protein interactions. In addition, alternative experimental and computational approaches are discussed to study aptamer-protein interactions.

    Mapping the sequences of potential guanine quadruplex motifs.

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    The knowledge that potential guanine quadruplex sequences (PQs) are non-randomly distributed in relation to genomic features is now well established. However, this is for a general potential quadruplex motif which is characterized by short runs of guanine separated by loop regions, regardless of the nature of the loop sequence. There have been no studies to date which map the distribution of PQs in terms of primary sequence or which categorize PQs. To this end, we have generated clusters of PQ sequence groups of various sizes and various degrees of similarity for the non-template strand of introns in the human genome. We started with 86 697 sequences, and successively merged them into groups based on sequence similarity, carrying out 66 clustering cycles before convergence. We have demonstrated here that by using complete linkage hierarchical agglomerative clustering such PQ sequence categorization can be achieved. Our results give an insight into sequence diversity and categories of PQ sequences which occur in human intronic regions. We also highlight a number of clusters for which interesting relationships among their members were immediately evident and other clusters whose members seem unrelated, illustrating, we believe, a distinct role for different sequence types

    G-Ruption: The Third International Meeting On G-Quadruplex And G-Assembly

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    A three and a half day conference focusing on nucleic acid structures called G-quadruplexes (G4s) and other guanine-based assemblies was held in Sorrento. Italy (June 28-July 1, 2011) and featured 35 invited talks and over 89 posters. The G-quadruplex field continues to expand at an explosive rate with the emergence of new connections to biology, chemistry, physics, and nanotechnology. Following the trend established by the previous two international G4 meetings, the conference touched upon all these areas and facilitated productive exchanges of ideas between researchers from all over the world
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