Role of the central cations in the mechanical unfolding of DNA and RNA G-quadruplexes.

Cations are known to mediate diverse interactions in nucleic acids duplexes but they are critical in the arrangement of four-stranded structures. Here, we use all-atom molecular dynamics simulations with explicit solvent to analyse the mechanical unfolding of representative intramolecular G-quadruplex structures: a parallel, a hybrid and an antiparallel DNA and a parallel RNA, in the presence of stabilising cations. We confirm the stability of these conformations in the presence of [Formula: see text] central ions and observe distortions from the tetrad topology in their absence. Force-induced unfolding dynamics is then investigated. We show that the unfolding events in the force-extension curves are concomitant to the loss of coordination between the central ions and the guanines of the G-quadruplex. We found lower ruptures forces for the parallel configuration with respect to the antiparallel one, while the behaviour of the force pattern of the parallel RNA appears similar to the parallel DNA. We anticipate that our results will be essential to interpret the fine structure rupture profiles in stretching assays at high resolution and will shed light on the mechanochemical activity of G-quadruplex-binding machinery

G-quadruplex formation of FXYD1 pre-mRNA indicates the possiblity of regulating expression of its protein product

G-quadruplexes are higher-order nucleic acid structures formed of square-planar arrangements of four guanine bases held together by Hoogsteen-type hydrogen bonds. Stacks of guanine tetrads are stabilised by intercalating potassium ions. FXYD1 encodes for phospholemman, a regulatory subunit of the cardiac Na+/K+-ATPase. Computational sequence analysis of FXYD1 pre-mRNA predicted the formation of stable intramolecular G-quadruplexes in human and orthologue sequences. Multiple sequence alignment indicated that G-rich sequences are conserved in evolution suggesting a potential role of G-quadruplexes in FXYD1 gene expression. The existence of a non-functional alternative splicing product indicated that the G-quadruplex formation may control alternative splicing. Quadruplex formation of human and bovine oligonucleotides was confirmed in vitro by native polyacrylamide gel electrophoresis and intrinsic fluorescence emission spectroscopy. Taking together the evolutionary conservation of G-quadruplex forming sequences with the confirmation of G-quadruplex formation in vitro by two FXYD1 homologues the results point to a potential role of these structures in regulating the expression of FXYD1 and thus regulate indirectly the activity of the cardiac Na+/K+ -ATPase.Peer reviewe

Formation of a Unique Cluster of G-Quadruplex Structures in the HIV-1 nef Coding Region: Implications for Antiviral Activity

G-quadruplexes are tetraplex structures of nucleic acids that can form in G-rich sequences. Their presence and functional role have been established in telomeres, oncogene promoters and coding regions of the human chromosome. In particular, they have been proposed to be directly involved in gene regulation at the level of transcription. Because the HIV-1 Nef protein is a fundamental factor for efficient viral replication, infectivity and pathogenesis in vitro and in vivo, we investigated G-quadruplex formation in the HIV-1 nef gene to assess the potential for viral inhibition through G-quadruplex stabilization. A comprehensive computational analysis of the nef coding region of available strains showed the presence of three conserved sequences that were uniquely clustered. Biophysical testing proved that G-quadruplex conformations were efficiently stabilized or induced by G-quadruplex ligands in all three sequences. Upon incubation with a G-quadruplex ligand, Nef expression was reduced in a reporter gene assay and Nef-dependent enhancement of HIV-1 infectivity was significantly repressed in an antiviral assay. These data constitute the first evidence of the possibility to regulate HIV-1 gene expression and infectivity through G-quadruplex targeting and therefore open a new avenue for viral treatment. © 2013 Perrone et al

Quadruplexes In ‘Dicty’: Crystal Structure Of A Four-Quartet G-Quadruplex Formed By G-Rich Motif Found In The Dictyostelium Discoideum Genome

Guanine-rich DNA has the potential to fold into non-canonical G-quadruplex (G4) structures. Analysis of the genome of the social amoeba Dictyostelium discoideum indicates a low number of sequences with G4-forming potential (249–1055). Therefore, D. discoideum is a perfect model organism to investigate the relationship between the presence of G4s and their biological functions. As a first step in this investigation, we crystallized the dGGGGGAGGGGTACAGGGGTACAGGGG sequence from the putative promoter region of two divergent genes in D. discoideum. According to the crystal structure, this sequence folds into a four-quartet intramolecular antiparallel G4 with two lateral and one diagonal loops. The G-quadruplex core is further stabilized by a G-C Watson–Crick base pair and a A–T–A triad and displays high thermal stability (Tm \u3e 90°C at 100 mM KCl). Biophysical characterization of the native sequence and loop mutants suggests that the DNA adopts the same structure in solution and in crystalline form, and that loop interactions are important for the G4 stability but not for its folding. Four-tetrad G4 structures are sparse. Thus, our work advances understanding of the structural diversity of G-quadruplexes and yields coordinates for in silico drug screening programs and G4 predictive tools

Ab initio RNA folding

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

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

Differential effect of alkali metal ions on the structure and stability of DNA G-Quadruplexes with potential role in the regulation of gene expression

G-quadruplexes (G-QPX) have been the subject of great interest in research due to their exclusive structural configuration, functionality and potential roles in biological applications particularly gene regulation and Nano-switch applications. Initial research has identified the presence of G-Quadruplex in the promoter regions of the human genome as well as untranslated regions of RNA. Promoter regions of human DNA have been found to be rich in Guanine which is one of the essential nucleotides required for the formation of G-Quadruplex structures with the potential to regulate gene activities, thereby acting as a biological switch by either allowing or disrupting the binding of transcription factors during gene expression. Ions such as potassium and sodium have been known to play key role in the formation and stability of G-Quadruplex leading to its potential role in up regulation and down regulation of transcription factors by interfering with their ability to bind on their specific binding sites on DNA. Though a variety of research has shown the potential role of alkali metal ions in the stability of quadruplex structure, not much has been done on the effect of concentration of theses ions on the structure of G-Quadruplex. The focus of this research is to understand the differential effect of alkali metal ions on the structure and stability of DNA G-Quadruplexes with potential role in the regulation of gene expression. Three alkali ions have been employed in this research, sodium, potassium ion because of their potential role in cell activities and Lithium ion because of its potential use in therapeutics. We also worked with four different modified crystal structures of the complex human telomeric repeat G-quadruplex (4fxm,1xav,217v and 2hri) In silico. The study was done under different ion concentration (50,100,150 and 160 mM). Different computational analysis was done on the structural change of GQuadruplex DNA with change in ion concentration using Gromacs computational software and algorithms. The obtained result suggests different response in structural change of G-Quadruplex DNA to changes in concentration of different ions. The effect of lithium ion on the structure of G-quadruplex has been an area of debate in the literature. Our results show that different G-Quadruplex and structures respond to variations in ion concentration differently, with some alkali ions favoring stability at lower concentration and acting differently at higher concentration. We observed deviations or structural changes in 4fxm G-Quadruplex with the three ions used in the simulation, sodium ion caused the most structural change, followed by lithium and potassium, making potassium more favorable in terms of structural change and stability. The same analysis was carried out on 1XAV G-Quadruplex, it also suggests lithium caused the most structural change followed by sodium and potassium, which further confirms potassium favors structural stability more than the other two ions (Li and Na+). A different result was observed on the structure of another G-Quadruplex 217v, the analysis shows a different response compared to the initial results, lithium caused the most structural change followed by potassium and sodium respectively. This could potentially tell that different G-Quadruplex structures may have a different response to variations in ions and ion concentration. Overall, potassium ion seems to favor GQuadruplex structural stability more than sodium and lithium. Lithium seem to cause more destabilizing effect at lower concentration and acts as stabilizing agent at higher concentration in some G-Quadruplex structures

In-line probing of RNA G-quadruplexes

Abstract: Although the majority of the initial G-quadruplex studies were performed on DNA molecules, there currently exists a rapidly growing interest in the investigation of those formed in RNA molecules that possess high potential of acting as gene expression regulatory elements. Indeed, G-quadruplexes found in the 50-untranslated regions of mRNAs have been reported to be widespread within the human transcriptome and to act as general translational repressors. In addition to translation regulation, several other mRNA maturation steps and events, including mRNA splicing, polyadenylation and localization, have been shown to be influenced by the presence of these RNA G-quadruplexes. Bioinformatic approaches have identified thousands of potential RNA G-quadruplex sequences in the human transcriptome. Clearly there is a need for the development of rapid, simple and informative techniques and methodologies with which the ability of these sequences, and of any potential new regulatory elements, to fold into G-quadruplexes in vitro can be examined. This report describes an integrated methodology for monitoring RNA G-quadruplexes formation that combines bioinformatic algorithms, secondary structure prediction, in-line probing with semi-quantification analysis and structural representation software. The power of this approach is illustrated, step-by-step, with the determination of the structure adopted by a potential G-quadruplex sequence found in the 50-untranslated region of the cAMP responsive element modulator (CREM) mRNA. The results unambiguously show that the CREM sequence folds into a G-quadruplex structure in the presence of a physiological concentration of potassium ions. This in-line probing-based method is easy to use, robust, reproducible and informative in the study of RNA G-quadruplex formation