Base Tetrads Research Articles

Probing telomeric-like G4 structures with full or partial 2′-deoxy-5-hydroxyuridine substitutions

Guanine quadruplexes (G4s) are stable four-stranded secondary DNA structures held together by noncanonical G-G base tetrads. We synthesised the nucleoside analogue 2′-deoxy-5-hydroxyuridine (H) and inserted its phosphoramidite into telomeric repeat-type model oligonucleotides. Full and partial substitutions were made, replacing all guanines in all the three tetrads of a three-tier G4 structure, or only in the putative upper, central, or lower tetrads. We characterised these modified structures using CD, UV absorbance spectroscopy, native gel studies, and a capture oligo-based G4 disruption kinetic assay. The strand separation activity of BLM helicase on these substituted structures was also investigated. Two of the partially H-substituted constructs adopted G4-like structures, but displayed lower thermal stabilities compared to unsubstituted G4. The construct modified in its central tetrad remained mostly denatured, but the possibility of a special structure for the fully replaced variant remained open. H substitutions did not interfere with the G4-resolving activity of BLM helicase, but its efficiency was highly influenced by construct topology and even more by the G4 ligand PhenDC3. Our results suggest that the H modification can be incorporated into G quadruplexes, but only at certain positions to maintain G4 stability. The destabilizing effect observed for 2′-deoxy-5-hydroxyuridine indicates that the cytosine deamination product 5-hydroxyuracil and its nucleoside counterpart in RNA (5-hydroxyuridine), might also be destabilizing in cellular DNA and RNA quadruplexes. The kinetic assay employed in this study can be generally employed for a fast comparison of the stabilities of various G4s either in their free or ligand-bound states.

Recognition of different base tetrads by RHAU (DHX36): X-ray crystal structure of the G4 recognition motif bound to the 3′-end tetrad of a DNA G-quadruplex

G-quadruplexes (G4) are secondary structures of nucleic acids that can form in cells and have diverse biological functions. Several biologically important proteins interact with G-quadruplexes, of which RHAU (or DHX36) – a helicase from the DEAH-box superfamily, was shown to bind and unwind G-quadruplexes efficiently. We report a X-ray co-crystal structure at 1.5 Å resolution of an N-terminal fragment of RHAU bound to an exposed tetrad of a parallel-stranded G-quadruplex. The RHAU peptide folds into an L-shaped α-helix, and binds to a G-quadruplex through π-stacking and electrostatic interactions. X-ray crystal structure of our complex identified key amino acid residues important for G-quadruplex-peptide binding interaction at the 3′-end G•G•G•G tetrad. Together with previous solution and crystal structures of RHAU bound to the 5′-end G•G•G•G and G•G•A•T tetrads, our crystal structure highlights the occurrence of a robust G-quadruplex recognition motif within RHAU that can adapt to different accessible tetrads.

Metal-Modified Nucleic Acids: Metal-Mediated Base Pairs, Triples, and Tetrads.

The incorporation of metal ions into nucleic acids by means of metal-mediated base pairs represents a promising and prominent strategy for the site-specific decoration of these self-assembling supramolecules with metal-based functionality. Over the past 20 years, numerous nucleoside surrogates have been introduced in this respect, broadening the metal scope by providing perfectly tailored metal-binding sites. More recently, artificial nucleosides derived from natural purine or pyrimidine bases have moved into the focus of AgI -mediated base pairing, due to their expected compatibility with regular Watson-Crick base pairs. This minireview summarizes these advances in metal-mediated base pairing but also includes further recent progress in the field. Moreover, it addresses other aspects of metal-modified nucleic acids, highlighting an expansion of the concept to metal-mediated base triples (in triple helices and three-way junctions) and metal-mediated base tetrads (in quadruplexes). For all types of metal-modified nucleic acids, proposed or accomplished applications are briefly mentioned, too.

Structural basis for activation of fluorogenic dyes by an RNA aptamer lacking a G-quadruplex motif

The DIR2s RNA aptamer, a second-generation, in-vitro selected binder to dimethylindole red (DIR), activates the fluorescence of cyanine dyes, DIR and oxazole thiazole blue (OTB), allowing detection of two well-resolved emission colors. Using Fab BL3-6 and its cognate hairpin as a crystallization module, we solved the crystal structures of both the apo and OTB-SO3 bound forms of DIR2s at 2.0 Å and 1.8 Å resolution, respectively. DIR2s adopts a compact, tuning fork-like architecture comprised of a helix and two short stem-loops oriented in parallel to create the ligand binding site through tertiary interactions. The OTB-SO3 fluorophore binds in a planar conformation to a claw-like structure formed by a purine base-triple, which provides a stacking platform for OTB-SO3, and an unpaired nucleotide, which partially caps the binding site from the top. The absence of a G-quartet or base tetrad makes the DIR2s aptamer unique among fluorogenic RNAs with known 3D structure.

Insights into G-quadruplex specific recognition by the DEAH-box helicase RHAU: Solution structure of a peptide–quadruplex complex

Four-stranded nucleic acid structures called G-quadruplexes have been associated with important cellular processes, which should require G-quadruplex-protein interaction. However, the structural basis for specific G-quadruplex recognition by proteins has not been understood. The DEAH (Asp-Glu-Ala-His) box RNA helicase associated with AU-rich element (RHAU) (also named DHX36 or G4R1) specifically binds to and resolves parallel-stranded G-quadruplexes. Here we identified an 18-amino acid G-quadruplex-binding domain of RHAU and determined the structure of this peptide bound to a parallel DNA G-quadruplex. Our structure explains how RHAU specifically recognizes parallel G-quadruplexes. The peptide covers a terminal guanine base tetrad (G-tetrad), and clamps the G-quadruplex using three-anchor-point electrostatic interactions between three positively charged amino acids and negatively charged phosphate groups. This binding mode is strikingly similar to that of most ligands selected for specific G-quadruplex targeting. Binding to an exposed G-tetrad represents a simple and efficient way to specifically target G-quadruplex structures.

Nucleic Acid Quadruplexes Based on 8-Halo-9-deazaxanthines: Energetics and Noncovalent Interactions in Quadruplex Stems

Structural and energetic features of artificial DNA quadruplexes consisting of base tetrads and their stacks with Na(+)/K(+) ion(s) inside the central pore and incorporating halogenated derivatives of xanthine, 8-fluoro-9-deazaxanthine (FdaX), 8-chloro-9-deazaxanthine (CldaX), 8-bromo-9-deazaxanthine (BrdaX), or 8-iodo-9-deazaxanthine (IdaX), have been investigated by modern state-of-the-art computational tools. The DNA (or RNA) quadruplex models based on 8-halo-9-deazaxanthines are predicted to be more stable relative to those with unmodified xanthine due to the increased stabilizing contributions coming from all three main types of weak interactions (H-bonding, stacking, and ion coordination). Methods for analyzing the electron density are used to understand the nature of forces determining the stability of the system and to gain a predictive potential. Quadruplex systems incorporating polarizable halogen atoms (chlorine, bromine, or iodine) benefit significantly from the stabilizing stacking between the individual tetrads due to an increased dispersion contribution as compared to xanthine and guanine, natural references used. Ion coordination induces a significant rearrangement of electron density in the quadruplex stem as visualized by electron deformation density (EDD) and analyzed by ETS-NOCV and Voronoi charges. Na(+) induces larger electron polarization from the quadruplex toward the ion, whereas K(+) has a higher propensity to electron sharing (identified by QTAIM delocalization index). We expect that our results will contribute to the development of novel strategies to further modify and analyze the natural G-quadruplex core.

Crystal Structure of an RNA Quadruplex Containing Inosine Tetrad: Implications for the Roles of NH 2 Group in Purine Tetrads

Polyinosinic acid has been known to adopt the four-stranded helical structure but its basic unit, inosine tetrad (I tetrad), has not been determined at the atomic level. Here we report the crystal structure of an RNA quadruplex containing an I tetrad at 1.4 Å resolution. The I tetrad has one cyclic hydrogen bond N1…O6 with the bond length of 2.7 Å. A water bridge is observed in the minor groove side of the base tetrad. Even though it is sandwiched by guanine tetrads (G tetrads), the I tetrad is buckled towards the 3′ side of the tetrad plane, which results from the different interaction strength with K ions on two sides of the tetrad plane. Comparison with both G tetrad and adenine tetrad indicates that lack of NH 2 in the C2 position makes the I tetrad prone to buckle for interactions with ligands. Two U*(G-G-G-G) base pentads are observed at the junction of the 5′ termini of two quadruplexes. The uridine residue in the base pentad is engaged in two hydrogen bonding interactions (N2(G)-H…O2(U) and O2′(G)-H…O4(U)) and a water-mediated interaction (N3(G) and N3(U)) with the G tetrad. We also discuss the roles of amino group in purine tetrads and the inter-quadruplex interactions in RNA molecules. These quadruplexes may interact with each other by stacking, groove binding and intercalation.

Base-tetrad swapping results in dimerization of RNA quadruplexes: implications for formation of the i-motif RNA octaplex.

Nucleic acids adopt different multistranded helical architectures to perform various biological functions. Here, we report a crystal structure of an RNA quadruplex containing "base-tetrad swapping" and bulged nucleotide at 2.1-Angstroms resolution. The base-tetrad swapping results in a dimer of quadruplexes with an intercalated octaplex fragment at the 5' end junction. The intercalated base tetrads provide the basic repeat unit for constructing a model of intercalated RNA octaplex. The model we obtained shows fundamentally different characteristics from duplex, triplex, and quadruplex. We also observed two different orientations of bulged uridine residues that are related to the interaction with surroundings. This structural evidence reflects the conformational flexibility of bulged nucleotides in RNA quadruplexes and implies the potential roles of bulged nucleotides as recognition and interaction sites in RNA-protein and RNA-RNA interactions.

Interaction of sodium and potassium ions with sandwiched cytosine‐, guanine‐, thymine‐, and uracil‐base tetrads

Nucleic acid tetraplexes and lipophilic self-assembling G-quadruplexes contain stacked base tetrads with intercalated metal ions as basic building blocks. Thus far, quantum-chemical studies have been used to explore the geometric and energetic properties of base tetrads with and without metal ions. Recently, for the first time, work on a sandwiched G-tetrad complex has been studied. We report here results of a systematic B3LYP density functional study on sandwiched G-, C-, U-, and T-tetrads with Na+ and K+ at different symmetries that substantially extend the recent work. The results include detailed information on total energies as well as on metal ion tetrad and base-base interaction energies. The geometrical parameters of the sandwiched metal ion complexes are compared to both experimental structures and to calculated geometries of complexes of single tetrads with metal ions. A microsolvation model explains the ion selectivity preference of K+ over Na+ in a qualitative sense.

H-bonding patterns in the platinated guanine-cytosine base pair and guanine-cytosine-guanine-cytosine base tetrad: an electron density deformation analysis and AIM study.

The atoms in molecule theory (AIM) and electronic structure analysis are applied together to investigate H-bonding patterns in metalated nucleobase complexes. The influence of Pt on the intra GC base pair H-bonding has been found to reduce intra base pair H-bonding of N4(C)...O6(G) in the platinated GC pair and GCGC tetrad. The relaxation of geometry constrains in metalated nucleobases is found to be decisively important in the formation of novel molecular architectures from nucleobases and metal entities. The incorporation of the platinum in the GCGC tetrad benefits the formation of the unique CH...N (H5(C)...N1(G)) hydrogen bond pattern in the tetrad by offering improved geometric constraints rather than through changing the electronic properties around the H5(C) and N1(G) sites. Platination at the N7 of guanine reduces the deprotonation energy considerably.

Feasibility of a two-metal, four-purine nucleobase quartet motif.

A mixed-purine nucleobase complex of composition trans-[(NH(3))(2)Pt(9-EtA-N1)(9-MeHx-N7)](NO(3))(2).2H(2)O (1) (9-EtA = 9-ethyladenine; 9-MeHx = 9-methylhypoxanthine) has been prepared and characterized by X-ray crystallography. Cations of 1 are self-complementary as far as hydrogen bonding properties are concerned and form H bonded dimers, containing four intermolecular hydrogen bonds in addition to two intramolecular ones. The resulting mixed-purine square is considered a model compound for a putative mixed-purine tetrad consisting of two adenines and two guanines. In this model, the one-metal, four-nucleobase quartet motif, as seen in guanine or uracil quartets of nucleic acids, with the metal located in the center of the base tetrad, has been altered to a two-metal, four-nucleobase motif, with the two metal ions localized at the periphery.

An Eight-Stranded Helical Fragment in RNA Crystal Structure: Implications for Tetraplex Interaction

Multistranded helical structures in nucleic acids play various functions in biological processes. Here we report the crystal structure of a hexamer, rU( BrdG)r(AGGU),at 1.5 Å resolution containing a structural complex of an alternating antiparallel eight-stranded helical fragment that is sandwiched in two tetraplexes. The octaplex is formed by groove binding interaction and base tetrad intercalation between two tetraplexes. Two different forms of octaplexes have been proposed, which display different properties in interaction with proteins and nucleic acids. Adenines form a base tetrad in the novel N6-H…N3 conformation and further interact with uridines to form an adenine-uridine octad in the reverse Hoogsteen pairing scheme. The conformational flexibility of adenine tetrad indicates that it can optimize its conformation in different interactions.

A novel form of intercalation involving four DNA duplexes in an acridine-4-carboxamide complex of d(CGTACG)(2).

The structures of the complexes formed between 9-amino-[N:-(2-dimethyl-amino)butyl]acridine-4-carboxamide and d(CG(5Br)UACG)(2) and d(CGTACG)(2) have been solved by X-ray crystallography using MAD phasing methodology and refined to a resolution of 1.6 A. The complexes crystallised in space group C222. An asymmetric unit in the brominated complex comprises two strands of DNA, one disordered drug molecule, two cobalt (II) ions and 19 water molecules (31 in the native complex). Asymmetric units in the native complex also contain a sodium ion. The structures exhibit novel features not previously observed in crystals of DNA/drug complexes. The DNA helices stack in continuous columns with their central 4 bp adopting a B-like motif. However, despite being a palindromic sequence, the terminal GC base pairs engage in quite different interactions. At one end of the duplex there is a CpG dinucleotide overlap modified by ligand intercalation and terminal cytosine exchange between symmetry-related duplexes. A novel intercalation complex is formed involving four DNA duplexes, four ligand molecules and two pairs of base tetrads. The other end of the DNA is frayed with the terminal guanine lying in the minor groove of the next duplex in the column. The structure is stabilised by guanine N7/cobalt (II) coordination. We discuss our findings with respect to the effects of packing forces on DNA crystal structure, and the potential effects of intercalating agents on biochemical processes involving DNA quadruplexes and strand exchanges. NDB accession numbers: DD0032 (brominated) and DD0033 (native).

Effect of Coordinated Ions on Structure and Flexibility of Parallel G-quadruplexes: A Molecular Dynamics Study

Single tract guanine residues can associate to form stable parallel quadruplex structures in the presence of certain cations. Nanosecond scale molecular dynamics simulations have been performed on fully solvated fibre model of parallel d(G7) quadruplex structures with Na+ or K+ ions coordinated in the cavity formed by the O6 atoms of the guanine bases. The AMBER 4.1 force field and Particle Mesh Ewald technique for electrostatic interactions have been used in all simulations. These quadruplex structures are stable during the simulation, with the middle four base tetrads showing root mean square deviation values between 0.5 to 0.8 Å from the initial structure as well the high resolution crystal structure. Even in the absence of any coordinated ion in the initial structure, the G-quadruplex structure remains intact throughout the simulation. During the 1.1 ns MD simulation, one Na+ counter ion from the solvent as well as several water molecules enter the central cavity to occupy the empty coordination sites within the parallel quadruplex and help stabilize the structure. Hydrogen bonding pattern depends on the nature of the coordinated ion, with the G-tetrad undergoing local structural variation to accommodate cations of different sizes. In the absence of any coordinated ion, due to strong mutual repulsion, O6 atoms within G-tetrad are forced farther apart from each other, which leads to a considerably different hydrogen bonding scheme within the G-tetrads and very favourable interaction energy between the guanine bases constituting a G-tetrad. However, a coordinated ion between G-tetrads provides extra stacking energy for the G-tetrads and makes the quadruplex structure more rigid. Na+ ions, within the quadruplex cavity, are more mobile than coordinated K+ ions. A number of hydrogen bonded water molecules are observed within the grooves of all quadruplex structures.

Modified oligonucleotides with a 5′-5′ interbase semi-rigid junction for alternate strand triplex formation

Solid-phase synthesis of 5′-5′-linked oligonucleotides with opposite polarities tethered via nucleic bases has been performed using a modified dinucleoside bearing an H-phosphonate group at the 3′-position of one nucleoside and a dimethoxytrityl group at the 3′-position of the second nucleoside. This system is aimed at forming a base tetrad at the junction in order to provide better stabilization. The linker used between the two 5′-terminal bases in the same plane involves a triple bond in order to rigidify the junction. The two oligonucleotide chains are either made of natural nucleosides or one of them is built with N3′ →P5′ phosphoramidates.

Bombyx mori single repeat telomeric DNA sequence forms a G-quadruplex capped by base triads.

A combined NMR-molecular dynamics approach has been applied to determine the solution structure of a truncated analogue of the Bombyx mori telomeric d(TTAGG) single repeat sequence in Na+ cation-containing aqueous solution. The two-fold symmetric four-stranded d(TAGG) quadruplex contains two adjacent G(syn).G(syn).G(anti).G(anti) G-tetrads sandwiched between novel (T.A).A triads with individual strands having both a parallel and antiparallel neighbour around the quadruplex. The (T.A).A triad represents the first experimental verification of a base triad alignment which constitutes a key postulate in the recently proposed model of triad-DNA. Further, the (T.A).A triad is generated by positioning an A residue through hydrogen bonding in the minor groove of a Watson-Crick T.A base pair and includes a T-A platform related to an A-A platform recently observed in the structure of the P4-P6 domain of the Tetrahymena self splicing group I ribozyme. The novel architecture of the truncated Bombyx mori quadruplex structure sets the stage for the design and potential identification of additional base tetrads and triads that could participate in pairing alignments of multi-stranded DNA structures during chromosome association and genetic recombination.

Modeling a strand exchange tetraplex conformation.

Molecular modeling had been used to study the conformation and the energetics of 4-stranded DNA complexes formed by strand exchange between two duplexes. Both isolated strand exchange tetraplexes (SET's) and duplex-tetraplex complexes are found to be stable. Hydrogen bonding between the major groove faces of the base pairs within each base tetrad is shown to be specific, allowing tetrad formation only between DNA duplexes having identical base sequences. Such structures can explain the recent experimental observations of Gaillard and Strauss concerning the complexation of two DNA containing poly(dCA) tracts and may be of relevance to genetic recombination mechanisms.

Evidence for interstrand quadruplex formation in the dimerization of human immunodeficiency virus 1 genomic RNA.

Retroviruses package two homologous single-stranded RNA genomes within a gag protein-RNA complex. In mature virion particles, the two RNA strands are thought to associate primarily through direct RNA-RNA interactions, although the structural basis for this stable association is unknown. We now report that a 127-nucleotide (nt) HIV-1NL4-3 RNA fragment (positions 732-858) encompassing the 5' end of the gag gene dimerizes spontaneously under high ionic strength conditions in the absence of any protein cofactor. The HIV-1 RNA dimer is dramatically and specifically stabilized by the monovalent cation potassium. Thermal dissociation of the dimer occurs at 80 degrees C in 100 mM K+ (5 mM Mg2+) but at significantly lower temperatures in the presence of either smaller or larger monovalent cations (100 mM Li+, 40 degrees C; 100 mM Na+, 55 degrees C; 100 mM Cs+, 30 degrees C). Deletion analyses of the 3' end of the 127-nt fragment reveal that an HIV-1 RNA fragment as short as 94 nt (732-825) can dimerize spontaneously, but a further 9-base deletion of the purine-rich sequence, GGGGGAGAA from positions 817 through 825, eliminates dimerization. These experimental results support a model in which HIV-1 RNA dimerizes by forming an interstrand quadruple helix stabilized by guanine (and/or purine)-base tetrads in analogy to the well-known dimerization of telomeric DNA. We speculate that this structure may also mediate the association of genomic HIV-1 RNA in vivo, revealing how RNA itself can achieve the self-recognition required for subsequent genetic recombination.

Telomeric DNA dimerizes by formation of guanine tetrads between hairpin loops.

The telomeric ends of eukaryotic chromosomes are composed of simple repeating sequences in which one DNA strand contains short tracts of guanine residues alternating with short tracts of A/T-rich sequences. The guanine-rich strand is always oriented in a 5'-3' direction towards the end of the chromosome and is extended to produce a 3' overhang of about two repeating units in species where the telomeric terminus is known. This overhang has been implicated in the formation of several unusual intra-and intermolecular DNA structures, although none of these structures has been characterized fully. We now report that oligonucleotides encoding Tetrahymena telomeres dimerize to form stable complexes in solution. This salt-dependent dimerization is mediated entirely by the 3'-terminal telomeric overhang (TT-GGGGTTGGGG) and produces complexes in which the N7 position of every guanine in the overhangs is chemically inaccessible. We therefore propose that telomeric DNA dimerizes by hydrogen bonding between two intramolecular hairpin loops, to form antiparallel quadruplexes containing cyclic guanine base tetrads. These novel hairpin dimers may be important in telomere association and recombination and could also provide a general mechanism for pairing two double helices in other recombinational processes.