Anh Tuân Phan

Human telomere, oncogenic promoter and 5′-UTR G-quadruplexes: diverse higher order DNA and RNA targets for cancer therapeutics

Guanine-rich DNA sequences can form G-quadruplexes stabilized by stacked G–G–G–G tetrads in monovalent cation-containing solution. The length and number of individual G-tracts and the length and sequence context of linker residues define the diverse topologies adopted by G-quadruplexes. The review highlights recent solution NMR-based G-quadruplex structures formed by the four-repeat human telomere in K+ solution and the guanine-rich strands of c-myc, c-kit and variant bcl-2 oncogenic promoters, as well as a bimolecular G-quadruplex that targets HIV-1 integrase. Such structure determinations have helped to identify unanticipated scaffolds such as interlocked G-quadruplexes, as well as novel topologies represented by double-chain-reversal and V-shaped loops, triads, mixed tetrads, adenine-mediated pentads and hexads and snap-back G-tetrad alignments. The review also highlights the recent identification of guanine-rich sequences positioned adjacent to translation start sites in 5′-untranslated regions (5′-UTRs) of RNA oncogenic sequences. The activity of the enzyme telomerase, which maintains telomere length, can be negatively regulated through G-quadruplex formation at telomeric ends. The review evaluates progress related to ongoing efforts to identify small molecule drugs that bind and stabilize distinct G-quadruplex scaffolds associated with telomeric and oncogenic sequences, and outlines progress towards identifying recognition principles based on several X-ray-based structures of ligand–G-quadruplex complexes.

Structural basis for gene regulation by a thiamine pyrophosphate-sensing riboswitch.

Riboswitches are metabolite-sensing RNAs, typically located in the non-coding portions of messenger RNAs, that control the synthesis of metabolite-related proteins. Here we describe a 2.05 Å crystal structure of a riboswitch domain from the Escherichia coli thiM mRNA that responds to the coenzyme thiamine pyrophosphate (TPP). TPP is an active form of vitamin B1, an essential participant in many protein-catalysed reactions. Organisms from all three domains of life, including bacteria, plants and fungi, use TPP-sensing riboswitches to control genes responsible for importing or synthesizing thiamine and its phosphorylated derivatives, making this riboswitch class the most widely distributed member of the metabolite-sensing RNA regulatory system. The structure reveals a complex folded RNA in which one subdomain forms an intercalation pocket for the 4-amino-5-hydroxymethyl-2-methylpyrimidine moiety of TPP, whereas another subdomain forms a wider pocket that uses bivalent metal ions and water molecules to make bridging contacts to the pyrophosphate moiety of the ligand. The two pockets are positioned to function as a molecular measuring device that recognizes TPP in an extended conformation. The central thiazole moiety is not recognized by the RNA, which explains why the antimicrobial compound pyrithiamine pyrophosphate targets this riboswitch and downregulates the expression of thiamine metabolic genes. Both the natural ligand and its drug-like analogue stabilize secondary and tertiary structure elements that are harnessed by the riboswitch to modulate the synthesis of the proteins coded by the mRNA. In addition, this structure provides insight into how folded RNAs can form precision binding pockets that rival those formed by protein genetic factors.

Human telomeric G‐quadruplex: structures of DNA and RNA sequences

Telomeres play an important role in cellular aging and cancer. Human telomeric DNA and RNA G‐rich sequences are capable of forming a four‐stranded structure, known as the G‐quadruplex. Such a structure might be important for telomere biology and a good target for drug design. This minireview describes the structural diversity or conservation of DNA and RNA human telomeric G‐quadruplexes, discusses structural views on targeting these G‐quadruplexes and presents some future challenges for structural studies.

Different loop arrangements of intramolecular human telomeric (3+1) G-quadruplexes in K+ solution

Intramolecular G-quadruplexes formed by the human telomeric G-rich strand are promising anticancer targets. Here we show that four-repeat human telomeric DNA sequences can adopt two different intramolecular G-quadruplex folds in K+ solution. The two structures contain the (3+1) G-tetrad core, in which three G-tracts are oriented in one direction and the fourth in the opposite direction, with one double-chain-reversal and two edgewise loops, but involve different loop arrangements. This result indicates the robustness of the (3+1) core G-quadruplex topology, thereby suggesting it as an important platform for structure-based drug design. Our data also support the view that multiple human telomeric G-quadruplex conformations coexist in K+ solution. Furthermore, even small changes to flanking sequences can perturb the equilibrium between different coexisting G-quadruplex forms.

Structural basis for Diels-Alder ribozyme-catalyzed carbon-carbon bond formation

The majority of structural efforts addressing RNAs catalytic function have focused on natural ribozymes, which catalyze phosphodiester transfer reactions. By contrast, little is known about how RNA catalyzes other types of chemical reactions. We report here the crystal structures of a ribozyme that catalyzes enantioselective carbon-carbon bond formation by the Diels-Alder reaction in the unbound state and in complex with a reaction product. The RNA adopts a λ-shaped nested pseudoknot architecture whose preformed hydrophobic pocket is precisely complementary in shape to the reaction product. RNA folding and product binding are dictated by extensive stacking and hydrogen bonding, whereas stereoselection is governed by the shape of the catalytic pocket. Catalysis is apparently achieved by a combination of proximity, complementarity and electronic effects. We observe structural parallels in the independently evolved catalytic pocket architectures for ribozyme- and antibody-catalyzed Diels-Alder carbon-carbon bond-forming reactions.

Structure-function studies of FMRP RGG peptide recognition of an RNA duplex-quadruplex junction

We have determined the solution structure of the complex between an arginine-glycine-rich RGG peptide from the human fragile X mental retardation protein (FMRP) and an in vitro–selected guanine-rich (G-rich) sc1 RNA. The bound RNA forms a newly discovered G-quadruplex separated from the flanking duplex stem by a mixed junctional tetrad. The RGG peptide is positioned along the major groove of the RNA duplex, with the G-quadruplex forcing a sharp turn of R10GGGGR15 at the duplex-quadruplex junction. Arg10 and Arg15 form cross-strand specificity–determining intermolecular hydrogen bonds with the major-groove edges of guanines of adjacent Watson-Crick G•C pairs. Filter-binding assays on RNA and peptide mutations identify and validate contributions of peptide-RNA intermolecular contacts and shape complementarity to molecular recognition. These findings on FMRP RGG domain recognition by a combination of G-quadruplex and surrounding RNA sequences have implications for the recognition of other genomic G-rich RNAs.

NMR spectroscopy of G-quadruplexes.

G-rich DNA and RNA sequences can form four-stranded structures called G-quadruplexes. Such structures have gained significant interest in the past decade with increasing evidence of their biological role. G-quadruplex structures can be polymorphic and dynamic. NMR spectroscopy has played an important role in G-quadruplex research. Here we review on the application of NMR techniques to study structure, dynamics and interaction of G-quadruplexes.

Bulges in G-Quadruplexes: Broadening the Definition of G-Quadruplex-Forming Sequences

We report on the first solution structure of an intramolecular G-quadruplex containing a single bulge and present evidence for extensive occurrence of bulges in different G-quadruplex contexts. The NMR solution structure of the d(TTGTGGTGGGTGGGTGGGT) sequence reveals a propeller-type parallel-stranded G-quadruplex containing three G-tetrad layers, three double-chain-reversal loops, and a bulge. All guanines participate in the formation of the G-tetrad core, despite the interruption between the first guanine and the next two guanines by a thymine, which forms a single-residue bulge and is projected out of the G-tetrad core. To provide a more general understanding about the formation of bulges within G-quadruplexes, we systematically investigated the effects of the residue type, the size, the position, and the number of bulges on the structure and stability of G-quadruplexes. The formation of bulges has also been observed in two different G-quadruplex scaffolds with different strand orientations and folding topologies. Our results show that bulges can be formed in many different situations within G-quadruplexes. While many sequences tested in this study can form stable G-quadruplex structures, all of them defy the description of sequences G3+NL1G3+NL2G3+NL3G3+, currently used in most bioinformatics searches for identifying potential G-quadruplex-forming sequences in the genomes. Broadening of this description to include the possibilities of bulge formation should allow the identification of more G-quadruplex-forming sequences which went unnoticed in the earlier searches. This study could also open the possibilities of exploiting bulges as recognition elements for interactions between G-quadruplexes and other molecules.

Stacking of G-quadruplexes: NMR structure of a G-rich oligonucleotide with potential anti-HIV and anticancer activity

G-rich oligonucleotides T30695 (or T30923), with the sequence of (GGGT)4, and T40214, with the sequence of (GGGC)4, have been reported to exhibit anti-HIV and anticancer activity. Here we report on the structure of a dimeric G-quadruplex adopted by a derivative of these sequences in K+ solution. It comprises two identical propeller-type parallel-stranded G-quadruplex subunits each containing three G-tetrad layers that are stacked via the 5′-5′ interface. We demonstrated control over the stacking of the two monomeric subunits by sequence modifications. Our analysis of possible structures at the stacking interface provides a general principle for stacking of G-quadruplexes, which could have implications for the assembly and recognition of higher-order G-quadruplex structures.

Solution structure of an intramolecular (3 + 1) human telomeric g-quadruplex bound to a telomestatin derivative.

Guanine-rich human telomeric DNA can adopt secondary structures known as G-quadruplexes, which can be targeted by small molecules to achieve anticancer effects. So far, the structural information on complexes between human telomeric DNA and ligands is limited to the parallel G-quadruplex conformation, despite the high structural polymorphism of human telomeric G-quadruplexes. No structure has been yet resolved for the complex with telomestatin, one of the most promising G-quadruplex-targeting anticancer drug candidates. Here we present the first high-resolution structure of the complex between an intramolecular (3 + 1) human telomeric G-quadruplex and a telomestatin derivative, the macrocyclic hexaoxazole L2H2-6M(2)OTD. This compound is observed to interact with the G-quadruplex through π-stacking and electrostatic interactions. This structural information provides a platform for the design of topology-specific G-quadruplex-targeting compounds and is valuable for the development of new potent anticancer drugs.