Fold-back tetraplex DNA species in DNase I-resistant DNA isolated from HeLa cells

A DNase I-resistant DNA species has been isolated and purified from HeLa cells by gel electrophoresis. Our studies indicate that the DNase I-resistant DNA species was about 40-60 bp fragment sizes responding to double-strand DNA marker and has higher guanine content. The image of AFM showed that this species has been assumed to be tetraplex structure according to its apparent width and height. Its CD, UV spectrum also exhibited characteristics similar to some tetraplex structure, which was different from the standard duplex DNA. 32P-labeled probes (TTAGGG)4 and 5'-TGGGGAGGGTGGGGAGGGTGGGGAAGG-3' could be hybridized to purified DNase I-resistant species. All results suggest that the DNase I-resistant DNA species have at least two components, which adopt an intrastrand fold-back DNA tetraplex. Their sequences were similar to human telomere and human c-myc locus (NHE), respectively.     

Suppression of c-myc oncogene expression by a polyamine-complexed triplex forming oligonucleotide in MCF-7 breast cancer cells.

Polyamines are excellent stabilizers of triplex DNA. Recent studies in our laboratory revealed a remarkable structural specificity of polyamines in the induction and stabilization of triplex DNA. 1,3-Diaminopropane (DAP) showed optimum efficacy amongst a series of synthetic diamines in stabilizing triplex DNA. To utilize the potential of this finding in developing an anti-gene strategy for breast cancer, we treated MCF-7 cells with a 37mer oligonucleotide to form triplex DNA in the up-stream regulatory region of the c-myc oncogene in the presence of DAP. As individual agents, the oligonucleotide and DAP did not downregulate c-myc mRNA in the presence of estradiol. Complexation of the oligonucleotide with 2 mM DAP reduced c-myc mRNA signal by 65% at 10 microM oligonucleotide concentration. In contrast, a control oligonucleotide had no significant effect on c-myc mRNA. The expression of c-fos oncogene was not significantly altered by the triplex forming oligonucleotide (TFO). DAP was internalized within 1 h of treatment; however, it had no significant effect on the level of natural polyamines. These data indicate that selective utilization of synthetic polyamines and TFOs might be an important strategy to develop anti-gene-based therapeutic modalities for breast cancer.     

DNA tetraplex formation studied with fluorescence resonance energy transfer.

It is emerging that DNA tetraplexes are pivotal for many major cellular processes, and techniques that assess their structure and nature to the point are under development. Here we show how the structural conversion of largely unstructured single-stranded DNA molecules into compact intrastrand DNA tetraplexes can be monitored by fluorescence resonance energy transfer. We recently reported that intrastrand tetraplex formation takes place in a nuclease hypersensitive element upstream of the human c-myc proto-oncogene. Despite the highly repetitive guanine-rich sequence of the hypersensitive element, fluorescence resonance energy transfer measurements indicate that only one well defined tetraplex structure forms therein. The proposed structure, which is specifically stabilized by potassium ions in vitro, has a core of three stacked guanine tetrads that is capped by two intrastrand A-T base pairs.     

Temperature and salt dependence of higher order structure formation by antisense c-myc and c-myb phosphorothioate oligodeoxyribonucleotides containing tetraguanylate tracts.

The use of complementary RNA or DNA sequences to selectively interfere with the utilization of mRNA of a target gene is an attractive therapeutic strategy. Two well-studied targets for oligonucleotide therapy are the c-mycand c-mybproto-oncogenes. It has been reported that sequences which contain four contiguous Gs can elicit a non-antisense response, due to the formation of a homotetrameric G quartet structure. Therefore, it was of interest to determine whether anti-c-mycand anti-c-mybphosphorothioate DNAs including tetraguanylate form higher order structures under physiologically relevant salt conditions and temperature. First, the identity of the higher order structure was established and was found to be a tetraplex. Employing intracellular (high K+), extracellular (low K+) and normal saline (no K+) salt mixtures, native gel electrophoresis revealed no tetraplex formation at 37 degrees C, the physiologically relevant temperature. On the other hand, tetraplex structure formation was observed at 4 and 23 degrees C. Hence, the potential for these sequences to form tetraplex structures at lower temperatures may not be relevant for their activity in cells and animals at physiological temperature.     

Tetraplex DNA transitions within the human c-myc promoter detected by multivariate curve resolution of fluorescence resonance energy transfer

The nuclease hypersensitive element (NHE) III(I) of the c-myc promoter regulates the expression of oncogene c-myc and hence is an important anti-cancer target. Paranemic secondary structure formation within the promoter has been implicated in mechanistic regulation models. Here, it is shown that two monomeric tetraplexes form within the c-myc promoter, which coexist in solution. The development and application of a new experimental approach for detection of conformation transitions in nucleic acids [which exploits the sensitivity of fluorescence resonance energy transfer (FRET) for theoretical spectral resolution by multivariate curve resolution-alternating least-squares (MCR-ALS) method] has been used for this study. The pK(a) for tetraplex transitions are centered around 5.9 +/- 0.2 (between two intercalation topologies) and 6.8 +/- 0.1 (tetraplex to random coil). The presence of two tetraplexes has been further confirmed by S1 nuclease digestion. Finally, it is established that MCR-ALS analysis of FRET at different temperatures, pH, and salt concentrations allows resolution of pure species. Results are discussed in the light of recent observations implicating paranemic DNA motifs within the c-myc NHE in regulation of the oncogene. This method has several advantages over other methods vis-à-vis, high sensitivity and linear detection over a wide concentration range and, particularly, potential applications in intracellular probing.     

Tetraplex formation of d(GGGGGTTTTT): 1H NMR study in solution.

The oligonucleotide d(G5T5) can in principle form a fully matched duplex with G.T pairing and/or a tetraplex. Non-denaturing gel electrophoresis, circular dichroism and NMR experiments show that the tetraplex is exclusively formed by this oligomer in solution. In the presence of its complementary strand d(A5C5) at low temperature, d(G5T5) forms the tetraplex over the normally expected Watson-Crick duplex. However, when d(G5T5) and d(A5C5) are mixed together in equimolar amounts and heated for several minutes at 85 degrees C, and then allowed to cool, the product was essentially the Watson-Crick duplex. The lack of resolution in the 500 MHz 1H NMR spectra and the presence of extensive spin diffusion do not allow us to derive a quantitative structure for the tetraplex from the NMR data. However, we find good qualitative agreement between the NOESY and MINSY data and a theoretically derived stereochemically sound structure in which the G's and T's are part of a parallel tetraplex.     

Quadruplex and pentaplex self-assemblies of oligonucleotides containing short runs of 8-aza-7-deaza-2'-deoxyisoguanosine or 2'-deoxyisoguanosine.

Oligonucleotides containing 8-aza-7-deaza-2'-deoxyisoguanosine (4) were investigated regarding their self-assembly in aqueous solution. The aggregation of 4 was compared with that of oligonucleotides containing 2'-deoxyisoguanosine (2b) and 2'-deoxyguanosine (1b). For this purpose the phosphoramidite of 4 was synthesized which was protected by a dibutylaminomethylidene residue at the amino group and a diphenylcarbamoyl residue at the 2-oxo function. Solid-phase synthesis furnished oligonucleotide containing short runs of the nucleoside 4. The self-assembly of the oligonucleotide 5'-d(T(4)4(4)T2) was studied by ion-exchange chromatography. The formation of a pentaplex was observed in the presence of Cs+, while a tetraplex is formed when the counter ion is Na+ or Rb+. The cation selectivity of the oligonucleotide 5'-d(T(4)4(4)T2) was found to be different from the parent 5'-d(T(4)isoG(4)T2) which was forming the tetraplex as well as a pentaplex in aq RbCl solution.     

Monovalent cation effects on intermolecular purine-purine-pyrimidine triple-helix formation.

The binding of a 19-mer guanosine-rich oligodeoxyribonucleotide, TG3TG4TG4TG3T (ODN 1), to a complementary polypurine DNA target was investigated by DNase I footprinting and restriction endonuclease protection assays. Monovalent cations inhibited intermolecular purine-purine-pyrimidine triple-helical DNA formation, with K+ and Rb+ being most effective, followed by NH4+ and Na+. Li+ and Cs+ had little to no effect. Similar results were observed with the G/A-rich oligonucleotide AG3AG4AG4AG3AGCT. Kinetic studies indicated that monovalent cations interfered with oligonucleotide-duplex DNA association but did not significantly promote triplex dissociation. The observed order of monovalent cation inhibition of triplex formation is reminiscent of their effect on tetraplex formation with G/T-rich oligonucleotides. However, using electrophoretic mobility shift assays we found that the oligonucleotide ODN 1 did not appear to form a four-stranded species under conditions promoting tetraplex formation. Taken together, our data suggest that processes other than the self-association of oligonucleotides into tetraplexes might be involved in the inhibitory effect of monovalent cations on purine-pyrimidine-purine triplex formation.     

Guanine tetraplex formation by short DNA fragments containing runs of guanine and cytosine.

Using CD spectroscopy, guanine tetraplex formation was studied with short DNA fragments in which cytosine residues were systematically added to runs of guanine either at the 5' or 3' ends. Potassium cations induced the G-tetraplex more easily with fragments having the guanine run at the 5' end, which is just an opposite tendency to what was reported for (G+T) oligonucleotides. However, the present (G+C) fragments simultaneously adopted other conformers that complicated the analysis. We demonstrate that repeated freezing/thawing, performed at low ionic strength, is a suitable method to exclusively stabilize the tetraplex in the (G+C) DNA fragments. In contrast to KCl, the repeated freeze/thaw cycles better stabilized the tetraplex with fragments having the guanine run on the 3' end. The tendency of guanine blocks to generate the tetraplex destabilized the d(G5).d(C5) duplex whose strands dissociated, giving rise to a stable tetraplex of (dG5) and single-stranded (dC5). In contrast to d(G3C3) and d(G5C5), repeated freezing/thawing induced the tetraplex even with the self-complementary d(C3G3) or d(C5G5); hence the latter oligonucleotides preferred the tetraplex to the apparently very stable duplex. The tetraplexes only included guanine blocks while the 5' end cytosines interfered neither with the tetraplex formation nor the tetraplex structure.     

Photoisomerizable arylstilbazolium ligands recognize parallel and antiparallel structures of G-quadruplexes

The photoisomerization and DNA interaction studies of three arylstilbazolium derivatives with various samples of nucleic acids (duplexes, triplexes and tetraplexes) are reported. The equilibrium dialysis study revealed high binding affinities of ligands to tetraplex structures. The quadruplex-binding affinity could be switched by light, e.g., the E,E and E,Z isomers of 1,4-bis(vinylquinolinium)benzene (1) interacted with parallel and antiparallel tetraplexes exhibiting different binding selectivity. The E,Z-1 showed higher binding preference for c-myc DNA (a propeller-type quadruplex), whereas the E,E-1 favorably interacted with telomeric DNA (a basket-type quadruplex). The presence of quadruplex DNA hampered photoisomerization of quadruplex-bound ligand.