Formation of the G-quadruplex and i-motif structures in retinoblastoma susceptibility genes (Rb)

The formation of G-quadruplex and i-motif structures in the 5′ end of the retinoblastoma (Rb) gene was examined using chemical modifications, circular dichroism (CD) and fluorescence spectroscopy. It was found that substitutions of 8-methylguanine at positions that show syn conformations in antiparallel G-quadruplexes stabilize the structure in the G-rich strand. The complementary C-rich 18mer forms an i-motif structure, as suggested by CD spectroscopy. Based on the C to T mutation experiments, C bases participated in the C–C⁺ base pair of the i-motif structure were determined. Experiments of 2-aminopurine (2-AP) substitution reveal that an increase of fluorescence in the G-quadruplex relative to duplex is attributed to unstacked 2-AP within the loop of G-quadruplex. The fluorescence experiments suggest that formation of the G-quadruplex and i-motif can compete with duplex formation. Furthermore, a polymerase arrest assay indicated that formation the G-quadruplex structure in the Rb gene acts as a barrier in DNA synthesis.     

Solution structure of the major G-quadruplex formed in the human VEGF promoter in K+: insights into loop interactions of the parallel G-quadruplexes

Vascular endothelial growth factor (VEGF) proximal promoter region contains a poly G/C-rich element that is essential for basal and inducible VEGF expression. The guanine-rich strand on this tract has been shown to form the DNA G-quadruplex structure, whose stabilization by small molecules can suppress VEGF expression. We report here the nuclear magnetic resonance structure of the major intramolecular G-quadruplex formed in this region in K⁺ solution using the 22mer VEGF promoter sequence with G-to-T mutations of two loop residues. Our results have unambiguously demonstrated that the major G-quadruplex formed in the VEGF promoter in K⁺ solution is a parallel-stranded structure with a 1:4:1 loop-size arrangement. A unique capping structure was shown to form in this 1:4:1 G-quadruplex. Parallel-stranded G-quadruplexes are commonly found in the human promoter sequences. The nuclear magnetic resonance structure of the major VEGF G-quadruplex shows that the 4-nt middle loop plays a central role for the specific capping structures and in stabilizing the most favored folding pattern. It is thus suggested that each parallel G-quadruplex likely adopts unique capping and loop structures by the specific middle loops and flanking segments, which together determine the overall structure and specific recognition sites of small molecules or proteins.LAY SUMMARY: The human VEGF is a key regulator of angiogenesis and plays an important role in tumor survival, growth and metastasis. VEGF overexpression is frequently found in a wide range of human tumors; the VEGF pathway has become an attractive target for cancer therapeutics. DNA G-quadruplexes have been shown to form in the proximal promoter region of VEGF and are amenable to small molecule drug targeting for VEGF suppression. The detailed molecular structure of the major VEGF promoter G-quadruplex reported here will provide an important basis for structure-based rational development of small molecule drugs targeting the VEGF G-quadruplex for gene suppression.     

Human telomeric DNA: G-quadruplex,i-motif and Watson-Crick double helix

Human telomeric DNA composed of (TTAGGG/CCCTAA)n repeats may form a classical Watson-Crick double helix. Each individual strand is also prone to quadruplex formation: the G-rich strand may adopt a G-quadruplex conformation involving G-quartets whereas the C-rich strand may fold into an i-motif based on intercalated C*C+ base pairs. Using an equimolar mixture of the telomeric oligonucleotides d[AGGG(TTAGGG)3] and d[(CCCTAA)3CCCT], we defined which structures existed and which would be the predominant species under a variety of experimental conditions. Under near-physiological conditions of pH, temperature and salt concentration, telomeric DNA was predominantly in a double-helix form. However, at lower pH values or higher temperatures, the G-quadruplex and/or the i-motif efficiently competed with the duplex. We also present kinetic and thermodynamic data for duplex association and for G-quadruplex/i-motif unfolding.     

Cationic porphyrins promote the formation of i-motif DNA and bind peripherally by a nonintercalative mechanism

Telomeric C-rich strands can form a noncanonical intercalated DNA structure known as an i-motif. We have studied the interactions of the cationic porphyrin 5,10,15,20-tetra-(N-methyl-4-pyridyl)porphine (TMPyP4) with the i-motif forms of several oligonucleotides containing telomeric sequences. TMPyP4 was found to promote the formation of the i-motif DNA structure. On the basis of (1)H NMR studies, we have created a model of the i-motif-TMPyP4 complex that is consistent with all the available experimental data. Two-dimensional NOESY data prompted us to conclude that TMPyP4 binds specifically to the edge of the intercalated DNA core by a nonintercalative mechanism. Since we have shown that TMPyP4 binds to and stabilizes the G-quadruplex form of the complementary G-rich telomeric strand, this study raises the intriguing possibility that TMPyP4 can trigger the formation of unusual DNA structures in both strands of the telomeres, which may in turn explain the recently documented biological effects of TMPyP4 in cancer cells.     

Evidence of the formation of G-quadruplex structures in the promoter region of the human vascular endothelial growth factor gene

The polypurine/polypyrimidine (pPu/pPy) tract of the human vascular endothelial growth factor (VEGF) gene is proposed to be structurally dynamic and to have potential to adopt non-B DNA structures. In the present study, we further provide evidence for the existence of the G-quadruplex structure within this tract both in vitro and in vivo using the dimethyl sulfate (DMS) footprinting technique and nucleolin as a structural probe specifically recognizing G-quadruplex structures. We observed that the overall reactivity of the guanine residues within this tract toward DMS was significantly reduced compared with other guanine residues of the flanking regions in both in vitro and in vivo footprinting experiments. We also demonstrated that nucleolin, which is known to bind to G-quadruplex structures, is able to bind specifically to the G-rich sequence of this region in negatively supercoiled DNA. Our chromatin immunoprecipitation analysis further revealed binding of nucleolin to the promoter region of the VEGF gene in vivo. Taken together, our results are in agreement with our hypothesis that secondary DNA structures, such as G-quadruplexes, can be formed in supercoiled duplex DNA and DNA in chromatin in vivo under physiological conditions similar to those formed in single-stranded DNA templates.     

Kinetic hybrid i-motifs: intercepting DNA with RNA to form a DNA(2)-RNA(2) i-motif

We have constructed and characterized a long-lived hybrid DNA(2)-RNA(2) i-motif that is kinetically formed by mixing equivalent amount of C-rich RNA (R) and C-rich DNA (D). Circular dichroism shows that these hybrids are distinct from their parent DNA(4) or RNA(4) i-motif. pH dependent CD and UV thermal melting experiments showed that the complexes were maximally stable at pH 4.5, the pK(a) of cytosine, consistent with the complex being held by CH(+)-C base pairs. Fluorescence studies confirmed their tetrameric nature and established the relative strand polarities of the RNA and DNA strands in the complex. These showed that in a hybrid D(2)R(2) i-motif two DNA strands occupy one narrow groove and the two RNA strands occupy the other. This suggests that even the sugar-sugar interactions are highly specific. Interestingly, this hybrid slowly disproportionates into DNA(4) i-motifs and ssRNA which would be valuable to study intermediates in DNA(4) i-motif formation.     

Orderly microaggregates of G-/C-rich oligonucleotides associated with spermine

Spermine-induced orderly assembling properties of G-/C-rich oligonucleotides are investigated in dilute and crowding conditions. The first time we report that the parallel G-quadruplexes is preferential to condense into anisotropic microaggregates in the presence of spermine, whereas the hybrid-type and the antiparallel G-quadruplexes have no significant interactions with spermine; and spermine can induce the condensation of i-motif C-rich oligonucleotides other than the random coiled C-rich strands. Moreover, the condensation of C-rich oligonucleotides can be reversibly regulated by pH and temperature. G-/C-rich oligonucleotides exhibit the cholesteric liquid crystalline phase at low strand concentration in the presence of spermine under crowding conditions. The results illuminate that the parallel G-quadruplex and i-motifs are probably necessity conformations for G-/C-rich oligonucleotides that involved in the regulation of chromosome organization in living cells.     

Tetraplex formation of surface-immobilized human telomere sequence probed by surface plasmon resonance using single-stranded DNA binding protein

Many sequences in genomic DNA are able to form unique tetraplex structures. Such structures are involved in a variety of important cellular processes and are emerging as a new class of therapeutic targets for cancers and other diseases. Screening for molecules targeting the tetraplex structure has been explored using such sequences immobilized on solid surfaces. Immobilized nucleic acids, in certain situations, may better resemble the molecules under in vivo conditions. In this report, we studied the formation of tetraplex structure of both the G-rich and C-rich strands of surface-immobilized human telomere sequence by surface plasmon resonance using the single-stranded DNA binding protein from Escherichia coli as probe. We demonstrate how the formation of G-quadruplex and i-motif could be probed under various conditions by this sequence-universal method. Our results also show that immobilization destabilized the tetraplex structure.     

Interactions of the human telomeric DNA with terbium-amino acid complexes

The human telomeric DNA can form four-stranded structures: the G-rich strand adopts a G-quadruplex conformation stabilized by G-quartets and the C-rich strand may fold into an I-motif based on intercalated C.C(+) base pairs. There is intense interests in the design and synthesis of compounds which can target telomeric DNA and inhibit the telomerase activity. Here we report the thermodynamic studies of the two newly synthesized terbium-amino acid complexes bound to the human telomeric G-quadruplex and I-motif DNA which were studied by means of UV-Visible, DNA meltings, fluorescence and circular dichroism. These two complexes can bind to the human telomeric DNA and have shown different features on DNA stability, binding stoichiometry, and sequence-dependent fluorescence enhancement. To our knowledge, this is the first report to show terbium-amino acid complexes can interact with the human telomeric DNA.     

G-quadruplex preferentially forms at the very 3' end of vertebrate telomeric DNA

Human chromosome ends are protected with kilobases repeats of TTAGGG. Telomere DNA shortens at replication. This shortening in most tumor cells is compensated by telomerase that adds telomere repeats to the 3′ end of the G-rich telomere strand. Four TTAGGG repeats can fold into G-quadruplex that is a poor substrate for telomerase. This property has been suggested to regulate telomerase activity in vivo and telomerase inhibition via G-quadruplex stabilization is considered a therapeutic strategy against cancer. Theoretically G-quadruplex can form anywhere along the long G-rich strand. Where G-quadruplex forms determines whether the 3′ telomere end is accessible to telomerase and may have implications in other functions telomere plays. We investigated G-quadruplex formation at different positions by DMS footprinting and exonuclease hydrolysis. We show that G-quadruplex preferentially forms at the very 3′ end than at internal positions. This property provides a molecular basis for telomerase inhibition by G-quadruplex formation. Moreover, it may also regulate those processes that depend on the structure of the very 3′ telomere end, for instance, the alternative lengthening of telomere mechanism, telomere T-loop formation, telomere end protection and the replication of bulky telomere DNA. Therefore, targeting telomere G-quadruplex may influence more telomere functions than simply inhibiting telomerase.     

Structure of a C-rich strand fragment of the human centromeric satellite III: a pH-dependent intercalation topology

Repetitive DNA sequences may adopt unusual pairing arrangements. At acid to neutral pH, cytidine-rich DNA oligodeoxynucleotides can form the i-motif structure in which two parallel-stranded duplexes with C.C(+) pairs are intercalated head-to-tail. The i-motif may be formed by multimeric associations or by intra-molecular folding, depending on the number of cytidine tracts, the nucleotide sequences between them, and the experimental conditions.We have found that a natural fragment of the human centromeric satellite III, d(CCATTCCATTCCTTTCC), can form two monomeric i-motif structures that differ in their intercalation topology and that are favored at pH values higher (the eta-form) and lower (the lambda-form) than 4.6. The change in intercalation may be related to adenine protonation in the loops.We studied the uridine derivative methylated on the first cytidine base, d(5mCCATTCCAUTCCUTTCC), whose proton spectrum is better resolved. The intercalation topologies are (C7.C17)/(5mC1.C11)/(C6.C16)/(C2.C12) for form lambda and (5mC1.C11)/(C7.C17)/(C2.C12)/(C6.C16) for form eta. We have solved the structure of the eta-form, and we present a model for the lambda-form. The switch from eta to lambda involves disruption of the i-motif. In both forms, the central AUT linker crosses the wide groove, and the first and the third linkers loop across the minor grooves. The i-motif core is extended in the eta-form by the inter-loop reverse Watson-Crick A3.U13 pair, whose dissociation constant is around 10(-2) at 0 degrees C, and in the lambda-form by the interloop T5.T15 pair.In contrast, d(5mCCATTCCTTACCTTTCC) folds into a pH-independent structure that has the same intercalation topology as the lambda-form. The i-motif core is extended below by the interloop T5.T15 pair and closed on top by the T8.A10 pair.Thus, the C-rich strand of the human satellite III tandem repeats, like the G-rich strand, can fold into various compact structures. The relevance of these features to centromeric function remains unknown.