Induction of duplex to G-quadruplex transition in the c-myc promoter region by a small molecule.

A major control element of the human c-myc oncogene is the nuclease-hypersensitive purine/pyrimidine-rich sequence. This double-stranded DNA fragment, corresponding to the 27-base pair segment in the nuclease-hypersensitive element of the c-myc promoter region, forms a stable Watson-Crick double helix under physiological conditions. However, this duplex DNA can be effectively converted to G-quadruplex DNA by a small molecular weight ligand. Both intermolecular and intramolecular G-quadruplex forms can be induced by this ligand. Similar transitional changes are also observed with the duplex telomeric sequence from the Oxytricha species. These results provide additional support to the idea that G-quadruplex structures may play structural roles in vivo and also provide insight into novel methodologies for rational drug design. These structurally altered DNA elements might serve as regulatory signals in gene expression or in telomere dynamics and hence are promising targets for drug action.

Design and synthesis of fluoroquinophenoxazines that interact with human telomeric G-quadruplexes and their biological effects.

In this study we have identified a new structural motif for a ligand with G-quadruplex interaction that results in biological effects associated with G-quadruplex-interactive compounds. Fluoroquinolones have been reported to possess weak telomerase inhibitory activity in addition to their better known bacterial gyrase poisoning. Starting with a fluoroquinobenzoxazine, which has modest potency in a human topoisomerase II assay, we have designed a more potent inhibitor of telomerase that has lost its topoisomerase II poisoning activity. This fluoroquinophenoxazine (FQP) interacts with G-quadruplex structures to inhibit the progression of Taq polymerase in a G-quadruplex polymerase stop assay. In addition, we demonstrate by 1H NMR studies that this compound interacts with telomeric G-quadruplex structures by external stacking to the G-tetrad with both the unimolecular fold-over and the parallel G-quadruplex structures. A photocleavage assay confirms the FQP interaction site, which is located off center of the external tetrad but within the loop region. Molecular modeling using simulated annealing was performed on the FQP-parallel G-quadruplex complex to determine the optimum FQP orientation and key molecular interactions with the telomeric G-quadruplex structure. On the basis of the results of these studies, two additional FQP analogues were synthesized, which were designed to test the importance of these key interactions. These analogues were evaluated in the Taq polymerase stop assay for G-quadruplex interaction. The data from this study and the biological evaluation of these three FQPs, using cytotoxicity and a sea urchin embryo system, were in accord with the predicted more potent telomeric G-quadruplex interactions of the initial lead compound and one of the analogues. On the basis of these structural and biological studies, the design of more potent and selective telomeric G-quadruplex-interactive compounds can be envisaged.

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.

G-quadruplexes as targets for drug design.

G-quadruplexes are a family of secondary DNA structures formed in the presence of monovalent cations that consist of four-stranded structures in which Hoogsteen base-pairing stabilizes G-tetrad structures. These structures are proposed to exist in vivo, although direct confirmatory evidence is lacking. Guanine-rich regions of DNA capable of forming G-quadruplex structures are found in a variety of chromosomal regions, including telomeres and promoter regions of DNA. In this review, we describe the design of three separate groups of G-quadruplex-interactive compounds and their interaction with G-quadruplex DNA. Using the first group of compounds (anthraquinones), we describe experiments that provide the proof of concept that a G-quadruplex is required for inhibition of telomerase. Using the second group of compounds (perylenes), we describe the structure of a G-quadruplex-ligand complex and its effect on the dynamics of formation and enzymatic unwinding of the quadruplex. For the third group of compounds (porphyrins), we describe the experiments that relate the biological effects to their interactions with G-quadruplexes.

NMR-Based model of a telomerase-inhibiting compound bound to G-quadruplex DNA.

The single-stranded (TTAGGG)n tail of human telomeric DNA is known to form stable G-quadruplex structures. Optimal telomerase activity requires the nonfolded single-stranded form of the primer, and stabilization of the G-quadruplex form is known to interfere with telomerase binding. We have identified 3,4,9, 10-perylenetetracarboxylic diimide-based ligands as potent inhibitors of human telomerase by using a primer extension assay that does not use PCR-based amplification of the telomerase primer extension products. A set of NMR titrations of the ligand into solutions of G-quadruplexes using various oligonucleotides related to human telomeric DNA showed strong and specific binding of the ligand to the G-quadruplex. The exchange rate between bound and free DNA forms is slow on the NMR time scale and allows the unequivocal determination of the binding site and mode of binding. In the case of the 5'-TTAGGG sequence, the ligand-DNA complex consists of two quadruplexes oriented in a tail-to-tail manner with the ligand sandwiched between terminal G4 planes. Longer telomeric sequences, such as TTAGGGTT, TTAGGGTTA, and TAGGGTTA, form 1:1 ligand-quadruplex complexes with the ligand bound at the GT step by a threading intercalation mode. On the basis of 2D NOESY data, a model of the latter complex has been derived that is consistent with the available experimental data. The determination of the solution structure of this telomerase inhibitor bound to telomeric quadruplex DNA should help in the design of new anticancer agents with a unique and novel mechanism of action.

A common 40 amino acid motif in eukaryotic RNases H1 and caulimovirus ORF VI proteins binds to duplex RNAs.

Eukaryotic RNases H from Saccharomyces cerevisiae , Schizosaccharomyces pombe and Crithidia fasciculata , unlike the related Escherichia coli RNase HI, contain a non-RNase H domain with a common motif. Previously we showed that S.cerevisiae RNase H1 binds to duplex RNAs (either RNA-DNA hybrids or double-stranded RNA) through a region related to the double-stranded RNA binding motif. A very similar amino acid sequence is present in caulimovirus ORF VI proteins. The hallmark of the RNase H/caulimovirus nucleic acid binding motif is a stretch of 40 amino acids with 11 highly conserved residues, seven of which are aromatic. Point mutations, insertions and deletions indicated that integrity of the motif is important for binding. However, additional amino acids are required because a minimal peptide containing the motif was disordered in solution and failed to bind to duplex RNAs, whereas a longer protein bound well. Schizosaccharomyces pombe RNase H1 also bound to duplex RNAs, as did proteins in which the S.cerevisiae RNase H1 binding motif was replaced by either the C.fasciculata or by the cauliflower mosaic virus ORF VI sequence. The similarity between the RNase H and the caulimovirus domain suggest a common interaction with duplex RNAs of these two different groups of proteins.

Solution structure of r(gaggacug):d(CAGTCCTC) hybrid: implications for the initiation of HIV-1 (+)-strand synthesis.

The three-dimensional solution structure of the hybrid duplex r(gaggacug):d(CAGTCCTC) has been determined by two-dimensional NMR, distance geometry (DG), restrained molecular dynamics (rMD) and NOE back-calculation methods. This hybrid, consisting of a purine-rich RNA strand and a pyrimidine-rich DNA strand, is related to the polypurine (+)-strand primer formed after (-)-strand DNA synthesis and RNase H degradation of the viral RNA strand and contains the site of a specific cleavage by reverse transcription (RT) RNase H at the end of the HIV-1 polypurine tract. This polypurine primer is an important intermediate in the formation of virally encoded double-stranded DNA prior to HIV-1 retrovirus integration. The correct processing of this primer is vital in the life cycle of the human immunodeficiency virus type (HIV-1) retrovirus. The structure of the r(gaggacug):d(CAGTCCTC) hybrid, as determined in solution by NMR, is intermediate between canonical A-type and B-type double helices, and has mixed structural characteristics. It is quantitatively different from the previously determined solution structures of other RNA-DNA hybrids, particularly in the width and shape of the major groove, which is wider than the major groove of other hybrids and is close to the dimension of the major groove of B-type DNA duplexes. The structure of this hybrid duplex contains a prominent bend in the double helix with a magnitude and direction similar to the bend in Okazaki fragments. The structural features of the present duplex may explain the unique interactions of this sequence with HIV-1 RT during both (-)-strand and (+)-strand DNA synthesis.

Structure of chimeric duplex junctions: solution conformation of the retroviral Okazaki-like fragment r(ccca)d(AATGA).d(TCATTTGGG) from Moloney murine leukemia virus.

We have determined the solution structure of the synthetic chimeric duplex r(ccca)d(AATGA).d(TCATTTGGG) by two-dimensional NMR, distance geometry, restrained molecular dynamics, and full relaxation matrix simulation of the two-dimensional nuclear Overhauser effect spectra at various mixing times. The chimeric strand of this duplex consists of the last four residues of the tRNA(Pro) primer for (-) strand DNA synthesis of Moloney murine leukemia virus and the first five residues of the (-) strand DNA produced by extending this primer; the complementary DNA strand corresponds to the (+) strand product from this template. The hybrid section of this chimeric duplex assumes a structure similar to that found for pure hybrid duplexes of mixed sequence, while the DNA section assumes a conformation closer to B-form DNA. There is significant distortion of the duplex at the hybrid-DNA junction which is manifested in marked changes in the helical parameters buckle, roll, and tip, changes in glycosidic torsion angles, and changes in the backbone torsion angles delta, epsilon, and zeta. The sugar conformations also undergo large changes, from heteromerous puckers in the hybrid section to a more B-form in the DNA section. Furthermore, the intrastrand phosphate separation in the chimeric strand is more typical of A-form duplexes in the RNA section but more like B-form duplexes in the DNA section. In the DNA section the minor groove width changes gradually from B-form at the periphery and approaches hybrid-like dimensions closer to the junction. The structural discontinuities act synergistically to produce a bend of 18 +/- 3 degrees at the junction. The global structure of this sequence is similar to that previously found in the chemically analogous Okazaki fragment r(gcg)d(TATACCC).d(GGGTATACGC) in solution. Such structure homology suggests a possible link between structure and function with respect to the recognition and cleavage of the junction RNA residues in both retroviral chimeras and Okazaki fragments during reverse transcription and normal DNA replication.

The DNA strand in DNA.RNA hybrid duplexes is neither B-form nor A-form in solution.

The structure of the DNA.RNA hybrid (GTCACATG).(caugugac), where lowercase letters designate RNA residues, has been determined on the basis of J-coupling analysis and 2D-NOE studies. The central hexamer in this sequence has been previously studied [Reid, D. G., Salisbury, S. A., Brown, T., Williams, D. H., Vasseur, J.-J., Rayner, B., & Imabach, J.-L. (1983) Eur. J. Biochem. 135, 307-314] via one-dimensional NOE methods and circular dichroism studies. Contrary to their results, we find that this duplex does not assume a B-form conformation in solution. Instead, the RNA residues retain their C3'-endo (A-form) conformation, as indicated by the absence of H1'-H2' couplings and by strong H6/H8 to (n-1) H2'NOEs. The sugars of the DNA residues, on the other hand, do not assume an A-form (or a B-form) conformation but an intermediate conformation in the O4'-endo range (P approximately 72-110 degrees), as indicated by the presence of strong H1'-H4' NOEs, medium-strength H2"-H3' COSY cross peaks, strong H3'-H4' DQF-COSY cross peaks, and H1'-H2' coupling constants that are of approximately the same magnitude as the H1'-H2" coupling constants. These results suggest that the RNA strand not only retains its N-type structure but also exerts an influence on the conformation of the DNA strand. Our results provide strong evidence that DNA.RNA hybrid duplexes do not assume an all-C2'-endo B-type conformation; neither do they assume an all-C3'-endo A-type conformation in solution. Furthermore, although not the main focus of this study, a comparison of the longitudinal relaxation times of the DNA and RNA residues indicates the need for extended relaxation delays in two-dimensional NMR spectra of hybrid duplexes, as has been previously observed for DNA.RNA chimeric duplexes (Wang, A. C., Kim, S.-G., Chou, S.-H., Orban, J., Flynn, P., & Reid, B. R. (1992) Biochemistry 31, 3940-3946).