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At least 703 records · Page 39Linked to original sources
PubMed2000

A novel inhibitor of human telomerase derived from 10H-indolo[3,2-b]quinoline.

The bis-dimethylaminoethyl derivative of quindoline (10H-indolo[3,2-b]quinoline), an alkaloid from the West African shrub Cryptolepis sanguinolenta, has been synthesised. This has been shown to have modest cytotoxicity, as well as inhibitory activity against the telomerase enzyme. It is hypothesised that the latter activity is due to stabilisation of an intermediate guanine-quadruplex complex, in accordance with computer modelling.

Aminoquinolines↗
PubMed1999

Human telomerase inhibition by substituted acridine derivatives.

A series of 3,6-disubstituted acridine derivatives have been rationally designed as telomerase inhibitors. They have been designed on the basis that inhibition of telomerase occurs by stabilising G-quadruplex structures formed by the folding of telomeric DNA. The most potent inhibitors have IC50 values against telomerase of between 1.3 and 8 microM, comparable to their cytotoxicity in ovarian cancer cell lines.

Acridines↗
PubMed1999

A ribozyme and a catalytic DNA with peroxidase activity: active sites versus cofactor-binding sites.

BACKGROUND: An 18-nucleotide DNA oligomer, PS2.M, derived using an in vitro selection method was previously reported to bind hemin (Fe(III)-protoporphyrinIX) with submicromolar affinity. The DNA-hemin complex exhibited DNA-enhanced peroxidative activity. PS2. M is guanine-rich and requires potassium ions to fold to its active conformation, consistent with its forming a guanine-quaduplex. In investigating the specific catalytic features of PS2.M we tested the peroxidative properties of its RNA version (rPS2.M) as well as that of an unrelated DNA guanine-quadruplex, OXY4. RESULTS: The hemin-binding affinity of rPS2.M was found to be 30-fold weaker than that of PS2.M. The UV-visible spectra and kinetics of enzymatic peroxidation of the RNA-hemin complex, however, were nearly identical to those of its DNA counterpart. Both displayed peroxidase activity substantially greater than those of heme proteins such as catalase and Fe(III)-myoglobin. Kinetic analysis suggested that PS2. M and rPS2.M catalyzed the breakdown of the hemin-hydrogen peroxide covalent complex to products. The hemin complex of folded OXY4 (which bound hemin as strongly as did rPS2.M) had a distinct absorption spectrum and only a minor peroxidase activity above the background level. CONCLUSIONS: The results indicated that it is possible for RNA and DNA of the same sequence to fold to form comparable cofactor-binding sites, and to show comparable catalytic behavior. The results further suggest that only a subset of cofactor-binding sites formed within folded nucleic acids might be able to function as active sites, by providing the appropriate chemical environments for catalysis.

Base Sequence↗
PubMed2003

The impact of nucleic acid secondary structure on PNA hybridization.

Hybridization of oligonucleotides and their analogues to complementary DNA or RNA sequences is complicated by the presence of secondary and tertiary structure in the target. In particular, folding of the target nucleic acid imposes substantial thermodynamic penalties to hybridization. Slower kinetics for hybridization can also be observed, relative to an unstructured target. The development of high affinity oligonucleotide analogues such as peptide nucleic acid (PNA) can compensate for the thermodynamic and kinetic barriers to hybridization. Examples of structured targets successfully hybridized by PNA oligomers include DNA duplexes, DNA hairpins, DNA quadruplexes and an RNA hairpin embedded within a mRNA.

Nucleic Acid Hybridization↗
PubMed2001

Telomerase: biology and phase I trials.

In normal somatic cells, the ends of chromosomes (the telomeres) shorten with each cell division. By contrast, in tumour cells, telomere length is maintained, generally through the reactivation of the reverse transcriptase enzyme, telomerase. At least three applications relating to telomeres and telomerase have been proposed: in cancer diagnosis and prognosis (especially through measurements of the catalytic component of telomerase, hTERT) and as a means of monitoring tumour response to therapy; as an aid to tissue engineering; and inhibition as a cancer therapeutic strategy. Mouse knockout, hTERT dominant negative, and antisense experiments suggest that telomerase inhibitors will confer anticancer activity, especially in tumours with short telomeres. Inhibitory strategies have focused on antisense molecules, inhibitors of reverse transcriptases, and small molecules able to interact with and stabilise four-stranded (G-quadruplex) structures formed by telomeres. Clinical trials involving telomerase inhibitors require careful consideration compared to those looking at conventional anticancer cytotoxic drugs.

Clinical Trials, Phase I as Topic↗
PubMed2003

Resolution of multiple ssDNA structures in free solution electrophoresis.

By using high concentrations of buffer, electroosmotic flow within uncoated channels of a microfluidic chip was minimized, allowing the free solution electrophoretic separation of DNA. More importantly, because of the ability to efficiently dissipate heat within these channels, field strengths as high as 600 V/cm could be applied with minimal Joule heating (<2 degrees C). As a result of the higher field strengths, separations within an 8-cm-long channel were achieved within a few minutes. However, when the electrophoretic separation of single-stranded DNA (ssDNA) less than 22 bases in length was performed, containing the fluorophore Texas Red as an end label, more than the expected single peak was observed at this high electric field. On the other hand, the free solution electrophoresis of a double-stranded DNA (dsDNA) consisting of a random sequence did exhibit the expected single peak. The appearance of these multiple peaks for ssDNA is shown to be dependent upon the base content and sequence of the ssDNA as well as on the chemical structure of the fluorophore used to tag the DNA for detection. Specifically, the peaks can be attributed to different secondary structures that result either from hydrophobic interactions between the DNA bases and an uncharged fluorescent dye or from G-quadruplexes within guanine-rich strands.

DNA, Single-Stranded↗
PubMed2002

Identifying hydrogen bond alignments in multistranded DNA architectures by NMR.

NMR studies of nucleic acids have benefited tremendously from the discovery of trans-hydrogen-bond scalar coupling constants, which have enabled direct determination of N-H...N and N-H...O=C hydrogen bonds using a combination of (2h)J(NN)-, (4h)J(NN)-, and (3h)J(NC)-based spectroscopy. This is especially true of multistranded DNA architectures containing intricate hydrogen-bonded networks mediated primarily through mismatched base pairing, which often resist identification by posing serious technical, spectroscopic, and physicochemical challenges. In this Account, we present a suite of NMR pulse sequences that have been developed in our laboratory to address these issues. We demonstrate the utility of these methods for identifying hydrogen bonds in two quadruplex DNA structures, containing triad, tetrad, and hexad motifs involving Watson-Crick, G.G and sheared G.A mismatch base pairing.

DNA↗
PubMed2000

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.

DNA↗
PubMed2000

Guanine specific binding at a DNA junction formed by d[CG(5-BrU)ACG](2) with a topoisomerase poison in the presence of Co(2+) ions.

The structure of the duplex d[CG(5-BrU)ACG](2) bound to 9-bromophenazine-4-carboxamide has been solved through MAD phasing at 2.0 A resolution. It shows an unexpected and previously unreported intercalation cavity stabilized by the drug and novel binding modes of Co(2+) ions at certain guanine N7 sites. For the intercalation cavity the terminal cytosine is rotated to pair with the guanine of a symmetry-related duplex to create a pseudo-Holliday junction geometry, with two such cavities linked through the minor groove interactions of the N2/N3 guanine sites at an angle of 40 degrees, creating a quadruplex-like structure. The mode of binding of the drug is shown to be disordered, with the major conformations showing the side chain bound to the N7 position of adjacent guanines. The other end of the duplex exhibits a terminal base fraying in the presence of Co(2+) ions linking symmetry-related guanines, causing the helices to intertwine through the minor groove. The stabilization of the structure by the intercalating drug shows that this class of compound may bind to DNA junctions as well as duplex DNA or to strand-nicked DNA ('hemi-intercalated'), as in the cleavable complex. This suggests a structural basis for the dual poisoning of topoisomerase I and II enzymes by this family of drugs.

Cations, Divalent↗
PubMed1994

A protein from Tetrahymena thermophila that specifically binds parallel-stranded G4-DNA.

G4-DNA is a parallel, four-stranded structure mediated by tetrads of hydrogen-bonded guanines (G-quartets). An abundant protein called Tetrahymena G4 binding protein (TGP) that binds to an intermolecular, quadruplex form of d(TTGGGGTTGGGGTTGGGGTTGGGG) under physiological salt conditions has been identified in cellular extracts from the ciliated protozoan Tetrahymena thermophila. In binding competition experiments, molecules capable of forming G4 structures compete for binding to TGP, but non-G4-forming molecules and r(U2G4)4 do not. TGP binding also requires a single-stranded region adjacent to the G4 structure. During the course of this study, it was determined that Mg2+ facilitates the formation of parallel-stranded G4-DNA structures and that high oligonucleotide concentrations are not required to drive formation of these structures. In addition, G4-DNA and TGP/G4-DNA complexes form readily under physiological salt conditions. These data support the proposal that G4-DNA structures exist in vivo.

Animals↗
PubMed2001

SELEX selection of high-affinity oligonucleotides for bacteriophage Ff gene 5 protein.

The Ff gene 5 protein (g5p) is a cooperative ssDNA-binding protein. SELEX was used to identify DNA sequences favorable for g5p binding at physiological ionic strength (200 mM NaCl) and 37 degrees C. Sequences were selected from a library of 58-mers that contained a central variable segment of 26 nucleotides. DNA sequences selected after eight rounds of SELEX were mostly G-rich, with multiple copies of CPuGGPy, TPuGGGPy, and/or PyPuPuGGGPy motifs. This was unexpected, since g5p has higher binding affinities for polypyrimidine than for polypurine sequences. The most recurrent G-rich sequence, named I-3, was found to have g5p-binding properties that were correlated with a structural transition. At 10 mM NaCl, I-3 existed in a single-stranded form that was saturated by g5p in an all-or-none fashion. At 200 mM NaCl, I-3 existed in a structured form that showed CD spectral features of G-quadruplexes. The g5p binding affinity for this structured form of I-3 was >100-fold higher than for the single-stranded form. Moreover, the structured I-3 was saturated by g5p in two steps, the first of which was the formation of an apparent initiation complex consisting of one I-3 strand and about three g5p dimers. Nuclease S1 footprinting and other experiments showed that g5p molecules in the initiation complex at 200 mM NaCl were bound directly to the G-rich variable segment and that the structure of I-3 was retained after saturation by g5p. Thus, G-rich motifs may form structures favorable for initiation of g5p binding and also provide the actual g5p-binding sites.

Bacteriophage M13↗
PubMed2002

Antiproliferative activity of G-quartet-forming oligonucleotides with backbone and sugar modifications.

Oligonucleotide-based therapies have considerable potential in cancer, viral, and cardiovascular disease therapies. However, it is becoming clear that the biological effects of oligonucleotides are not solely due to the intended sequence-specific interactions with nucleic acids. Oligonucleotides are also capable of interacting with numerous cellular proteins owing to their polyanionic character or specific secondary structure. We have examined the antiproliferative activity, protein binding, and G-quartet formation of a series of guanosine-rich oligonucleotides, which are analogues of GRO29A, a G-quartet forming, growth-inhibitory oligonucleotide, whose effects we have previously described [Bates P. J., Kahlon, J. B., Thomas, S. D., Trent, J. O., and Miller, D. M. (1999) J. Biol. Chem. 274, 26369-26377]. The GRO29A analogues include phosphorothioate (PS29A), 2'-O-methyl RNA (MR29A), and mixed DNA/2'-O-methyl RNA (MRdG29A) oligonucleotides. We demonstrate by UV spectroscopy that all of the modified analogues form stable structures, which are consistent with G-quartet formation. We find that the phosphorothioate and mixed DNA/2'-O-methyl analogues are able to significantly inhibit proliferation in a number of tumor cell lines, while the 2'-O-methyl RNA has no significant effects. Similar to the original oligonucleotide, GRO29A, the growth inhibitory oligonucleotides were able to compete with the human telomere sequence oligonucleotide for binding to a specific cellular protein. The less active MR29A does not compete significantly for this protein. On the basis of molecular modeling of the oligonucleotide structures, it is likely that the inactivity of MR29A is due to the differences in the groove structure of the quadruplex formed by this oligonucleotide. Interestingly, all GRO29A analogues, including an unmodified DNA phosphodiester oligonucleotide, are remarkably resistant to nuclease degradation in the presence of serum-containing medium, indicating that secondary structure plays an important role in biological stability. The remarkable stability and strong antiproliferative activity of these oligonucleotides confirm their potential as therapeutic agents.

Base Sequence↗
PubMed2004

Targeting DNA with novel diphenylcarbazoles.

Double-stranded DNA is a therapeutic target for a variety of anticancer and antimicrobial drugs. Noncovalent interactions of small molecules with DNA usually occur via intercalation of planar compounds between adjacent base pairs or minor-groove recognition by extended crescent-shaped ligands. However, the dynamic and flexibility of the DNA platform provide a variety of conformations that can be targeted by structurally diverse compounds. Here, we propose a novel DNA-binding template for construction of new therapeutic candidates. Four bisphenylcarbazole derivatives, derived from the combined molecular architectures of known antitumor bisphenylbenzimidazoles and anti-infectious dicationic carbazoles, have been designed, and their interaction with DNA has been studied by a combination of biochemical and biophysical methods. The substitutions of the bisphenylcarbazole core with two terminal dimethylaminoalkoxy side chains strongly promote the interaction with DNA, to prevent the heat denaturation of the double helix. The deletion or the replacement of the dimethylamino-terminal groups with hydroxyl groups strongly decreased DNA interaction, and the addition of a third cationic side chain on the carbazole nitrogen reinforced the affinity of the compound for DNA. Although the bi- and tridentate molecules both derive from well-characterized DNA minor-groove binders, the analysis of their binding mode by means of circular and linear dichroism methods suggests that these compounds form intercalation complexes with DNA. Negative-reduced dichroism signals were recorded in the presence of natural DNA and synthetic AT and GC polynucleotides. The intercalation hypothesis was validated by unwinding experiments using topoisomerase I. Prominent gel shifts were observed with the di- and trisubstituted bisphenylcarbazoles but not with the uncharged analogues. These observations, together with the documented stacking properties of such molecules (components for liquid crystals), prompted us to investigate their binding to the human telomeric DNA sequence by means of biosensor surface plasmon resonance. Under conditions favorable to G4 formation, the title compounds showed only a modest interaction with the telomeric quadruplex sequence, comparable to that measured with a double-stranded oligonucleotide. Their sequence preference was explored by DNase I footprinting experiments from which we identified a composite set of binding sequences comprising short AT stretches and a few other mixed AT/GC blocks with no special AT character. The variety of the binding sequences possibly reflects the coexistence of distinct positioning of the chromophore in the intercalation sites. The bisphenylcarbazole unit represents an original pharmacophore for DNA recognition. Its branched structure, with two or three arms suitable to introduce a structural diversity, provides an interesting scaffold to built molecules susceptible to discriminate between the different conformations of nucleic acids.

Base Sequence↗
PubMed2004

Structural dynamics and processing of nucleic acids revealed by single-molecule spectroscopy.

Single-molecule fluorescence spectroscopy is a powerful method to observe real time movements of individual biological molecules while they are functioning without the need for synchronization. Dynamic characteristics of nucleic acids can now be easily and reliably studied, and new applications are emerging in which their recognition and processing by proteins and enzymes are being understood with unprecedented detail. The most recent examples are discussed, including the hairpin ribozyme, Holliday junction, G-quadruplex, Rep helicase, reverse transcriptase, and combination with mechanical manipulation.

DNA↗
PubMed1998

Characterization of purine-motif triplex DNA-binding proteins in HeLa extracts.

One piece of evidence indicating that triple-helical DNAs exist in vivo would be the demonstration of cellular proteins that recognize such structures. Using oligonucleotide probes containing a GT-rich purine-motif triplex, proteins from either HeLa nuclear or cytoplasmic extracts and electrophoretic mobility shift assays, we identified four specific human protein-triplex complexes. Proteins in these complexes did not recognize an analogous homopurine/homopyrimidine duplex DNA or a pyrimidine-motif triplex but did recognize purine-motif triplexes regardless of whether they possessed a phosphodiester or phosphorothioate backbone in the third strand or involved A*AT instead of T*AT base triplets. For each of these proteins, binding affinity increased with increasing triplex length. For some triplex-binding proteins, a weak affinity was noted for individual G-rich oligonucleotides, though this may actually reflect an affinity for quadruplex structures, which these oligonucleotides are prone to adopt. Ion exchange chromatographic fractionation of HeLa nuclear extracts indicated that at least three different proteins were responsible for the observed electrophoretic mobility shifts. Southwestern blotting methods identified three major polypeptides, with apparent molecular masses of 100, 60, and 15 kDa, that preferentially recognized purine-motif triplexes. These data demonstrate the existence of eukaryotic proteins that specifically recognize one triplex motif and support the idea of a biological role for triple helical DNA.

Binding, Competitive↗
PubMed1999

Regulation of catalytic activity and processivity of human telomerase.

The ends of eukaryotic chromosomes are specialized sequences, called telomeres comprising tandem repeats of simple DNA sequences. Those sequences are essential for preventing aberrant recombination and protecting genomic DNA against exonucleolytic DNA degradation. Telomeres are maintained at a stable length by telomerase, an RNA-dependent DNA polymerase. Recently, human telomerase has been recognized as a unique diagnostic marker for human tumors and is potentially a highly selective target for antitumor drugs. In this study, we have examined the major factors affecting the catalytic activity and processivity of human telomerase. Specifically, both the catalytic activity and processivity of human telomerase were modulated by temperature, substrate (dNTP and primer) concentration, and the concentration of K+. The catalytic activity of telomerase increased as temperature (up to 37 degrees C), concentrations of dGTP, primer, and K+ were increased. However, the processivity of human telomerase decreased as temperature, primer concentration, and K+ were increased. Our results support the current model for human telomerase reaction and strengthen the hypothesis that a G-quadruplex structure of telomere DNA plays an important role in the regulation of the telomerase reaction.

Catalysis↗
PubMed2000

Effect of loop sequence and size on DNA aptamer stability.

The thrombin aptamer is a 15-mer oligodeoxyribonucleotide that folds into a unimolecular quadruplex consisting of a stack of two guanine quartets connected by two external loops and one central loop and possesses a high affinity for thrombin. We have undertaken a systematic examination, in KCl, of the thermodynamic stability of thrombin aptamer analogues containing sequence modifications in one or more of the loops, as well as in the number of quartets. UV melting studies have been carried out to obtain the relevant thermodynamic parameters for these aptamers. van't Hoff analysis of these data, with a two-state model for unimolecular denaturation, gave excellent fits to the experimental observations. Thermodynamic analysis indicates that the central loop sequence in the parent aptamer is optimal for stability. Modifications in this or other loops can effect either DeltaH degrees, DeltaS degrees, or both. Addition of a single G at the 5'-end decreases stability while addition of a G at the 3'-end increases stability. Differential scanning calorimetry experiments on the thrombin aptamer reveal that a heat capacity change, not detected by UV measurements, accompanies the unfolding of the aptamer.

Calorimetry, Differential Scanning↗
PubMed2002

A site-specific low-enrichment (15)N,(13)C isotope-labeling approach to unambiguous NMR spectral assignments in nucleic acids.

We demonstrate that base and sugar protons within a DNA oligomer can be assigned unambiguously following site-specific 15N,13C isotope incorporation at levels as low as 1% enrichment. This simple and cost-effective methodology is demonstrated on the d(GGGTTCAGG) DNA sequence, which forms a dimeric G-quadruplex containing G.G.G.G tetrads sandwiched between G.(C-A) triads.

Carbon Isotopes↗