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.     

Condensation reaction of hexanucleotides containing guanine and cytosine with water soluble carbodiimide.

The condensation reactions of hexanucleotides involving guanine and cytosine in the presence of water-soluble carbodiimide (WSC) have been investigated as a model reaction of the prebiotic formation of RNA under primitive earth. The reactions formed cyclic hexanucleotides and dimers in which the product yields were dependent on the sequence.     

Sequence dependence of conformations of self-complementary duplex tetradeoxynucleotides containing cytosine and guanine

The four self-complementary tetradeoxynucleotides which contain only cytosine and guanine are 5'-d-(CpGpCpG)-3', 5'-d(CpCpGpG)-3', 5'-d(GpCpGpC)-3', and 5'-d(GpGpCpC)-3'. The Raman spectra of aqueous solutions (about 0.05 M in monomer) of these tetranucleotides at pH 7 and 2 degrees C show clearly that these self-complementary tetranucleotides form double-stranded duplex structures of the canonical B type when the NaCl concentration is 0.5 M NaCl. If the temperature is raised to 50 degrees C, the Raman spectra show that in each case the double-helical B form melts in a non-cooperative way to a disordered single-chain form. On the other hand, if the salt concentration is raised to saturation, the Raman spectrum of only one of these four tetranucleotide solutions at 2 degrees C is changed in any substantial way. The Raman spectrum of the tetranucleotide 5'-d(CpGpCpG)-3' at 2.2 degrees C and at 4 M or higher salt concentration strongly resembles that of double-helical Z-form poly(dC-dG) taken under similar conditions. We conclude that the tetramer 5'-d(CpGpCpG)-3' is the only self-complementary double-helical tetranucleotide containing only cytosine and guanine in which the B-Z transition can be induced by increasing the salt concentration. This tetramer has several types of stacking interactions which differ markedly from stacking interactions in the other tetramers and may account for the enhanced stabilization of its Z conformation.     

Interaction of the dye ethidium bromide with DNA containing guanine repeats.

DNA containing one or more copies of the motifs repeated in telomere sequences has unusual conformational properties. The isolated sequence from the protozoan Oxytricha, dT4G4 has the potential to form tetramers in the presence of sodium or potassium ions. We report here that these tetramers bind ethidium tightly, with an interaction that fulfills several criteria for an intercalative mechanism in the G sequence. By contrast, the 4-fold tandem repeat of this subunit, d(T4G4)4, does not interact specifically with ethidium in the presence of Na+. This difference might have a simple structural basis: the tetramer of dT4G4 forms a stack of four G-quartets in the presence of Na+ or K+, whereas the constraint imposed by the T4 "tethers" in the repeat d(T4G4)4 allows only two layers to form in the presence of Na+. In the presence of sufficient K+, the latter can partially form a four-layer G-quartet structure, which interacts with ethidium. This idea is supported by analysis of a "relaxed" sequence, dT4G4(T7G4)3, which allows formation of four G-quartets and binds ethidium in the presence of Na+ as well as K+. Ethidium (and intercalators generally) should thus be able to retard or inhibit the action of telomerase in the presence of K+.     

Inhibitory short synthetic oligodeoxynucleotides and lupus

B cells and antigen-presenting cells express a group of intracellular Toll-like receptors (TLRs) that recognize nucleic acids and can be accessed only when apoptotic debris or immune complexes are internalized by B-cell receptors or Fc receptors. This results in rapid cell activation and release of inflammatory mediators that perpetuate the autoantibody response. TLR-7 and TLR-9 are required to generate autoantibodies to RNA and DNA, respectively. Synthetic oligodeoxynucleotides that inhibit the activity of these intracellular TLRs attenuate systemic lupus erythematosus in mouse models and may be of therapeutic benefit in human systemic lupus erythematosus.     

N-Sulfomethylation of guanine, adenine and cytosine with formaldehyde-bisulfite. A selective modification of guanine in DNA.

When guanine-, adenine- and cytosine-nucleosides and nucleotides were treated with formaldehyde and then with bisulfite, stable N-sulfomethyl compounds were formed. N2-Sulfomethylguanine, N6-sulfomethyladenine, N4-sulfomthylcytosine and N6-sulfomethyl-9-beta-D-arabinofuranosyladenine were isolated as crystals and characterized. A guanine-specific sulfomethylation was brought about by treatment and denatured single-stranded DNA with formaldehyde and then with bisulfite at pH 7 and 4 degrees C. Since native double-stranded DNA was not modified by this treatment, this new method of modification is expected to be useful as a conformational probe for polynucleotides.     

Reactivity of mitomycin C with synthetic polyribonucleotides containing guanine or guanine analogs

The guanine residues in nucleic acids are believed to be the major covalent binding site of the antibiotic mitomycin C. To identify the specific functional group in guanine which reacts with mitomycin C, reactions were run between the antibiotic and poly(G) analogs in which guanine was blocked at the N-7 or O-6 position, or lacked the 2-amino group. Binding ratios were affected to a small extent in the two former cases, but binding was significantly decreased in the absence of the 2-amino group. These results indicate that the most likely binding site of mitomycin C in synthetic polyribonucleotides is the 2-amino group of guanine residues.     

Binding of transition metal complexes to guanine and guanine–cytosine: hydrogen bonding and covalent effects

Density functional calculations and Atoms in Molecules analysis are used to investigate the role of covalent and hydrogen bondings in determining the binding of transition metal complexes to guanine, and the subsequent effect on pairing with cytosine. Hydrogen bonding is ubiquitous, and typically contributes ca. 10% to overall binding, a value that varies with the coordination site on guanine, as well as metal and ligands. Early transition metals show a clear preference for the O6 position, while later ones prefer N7, the crossover point coming at the vanadium group. Metallation at N7 causes a redistribution of hydrogen bonding strength between guanine and cytosine, but does not greatly affect the overall pairing energy. In contrast, metallation at O6 strongly reduces the pairing energy, as may be expected given the role of O6 in pairing guanine with cytosine. This effect can be quantified using electron density properties, and seems to be due to both electrostatic repulsion from the positive metal centre and a redistribution of electron density within guanine itself. Qualitative agreement with experimental mass spectroscopic results is obtained.     

Properties of pseudo-complementary DNA substituted with weakly pairing analogs of guanine or cytosine

A straightforward enzymatic protocol for converting regular DNA into pseudo-complementary DNA could improve the performance of oligonucleotide microarrays by generating readily hybridizable structure-free targets. Here we screened several highly destabilizing analogs of G and C for one that could be used with 2-aminoadenine (nA) and 2-thiothymine (sT) to generate structure-free DNA that is fully accessible to complementary probes. The analogs, which included bioactive bases such as 6-thioguanine (sG), 5-nitrocytosine (NitroC), 2-pyrimidinone (P; the free base of zebularine) and 6-methylfuranopyrimidinone (MefP), were prepared as dNTPs and evaluated as substrates for T7 and Phi29 DNA polymerases that lacked editor function. Pairing properties of the analogs were characterized by solution hybridization assays using modified oligonucleotides or primer extension products. P and MeP did not support robust primer extension whereas sG and NitroC did. In hybridization assays, however, sG lacked discrimination and NitroC paired too strongly to C. The dNTPs of two other base analogs, 7-nitro-7-deazahypoxanthine (NitrocH) and 2-thiocytosine (sC), exhibited the greatest promise. Either analog could be used with nA and sT to generate DNA that was nearly structure-free. Hybridization of probes to these modified DNAs will require the development of base analogs that pair strongly to NitrocH or sC.     

Interaction of echinomycin with guanine: electrochemistry and spectroscopy studies

The interaction of antitumor antibiotic, echinomycin (Echi) with guanine (Gua) was thoroughly investigated by adsorptive transfer stripping cyclic voltammetry, ultraviolet and visible adsorption spectra (UV/Vis) and Fourier-transform infrared spectroscopy (FTIR). Electrochemistry provided a simple tool for verifying the occurrence of interaction between Echi and Gua. Echi could be accumulated from the solution and give well-defined electrochemical signals in 0.1 M phosphate buffer solution (pH 7.0) only when Gua was present on the surface of the electrochemically pretreated glass carbon electrode (GCE), suggesting a strong binding of Echi to Gua. All the acquired spectral data showed that a new adduct between Echi and Gua was formed, and two pairs of adjacent intermolecular hydrogen bonds between the Ala backbone atoms in Echi and Gua (Ala-NH to Gua-N3 and Gua-NH2 to Ala-CO) played a dominating role in the interaction. Electrochemistry coupled with spectroscopy techniques could provide a relatively easy way to obtain useful insights into the molecular mechanism of drug-DNA interactions, which should be important in the development of new anticancer drugs with specific base recognition.     

Treatment of infectious diseases with immunostimulatory oligodeoxynucleotides containing CpG motifs

Bacterial DNA with CpG motifs can efficiently stimulate the vertebrate immune system. Thus, synthetic oligodeoxynucleotides that contain such CpG motifs (CpG-ODN) are currently used in preclinical and clinical studies to develop new allergy or cancer therapies and vaccine adjuvants. Recent animal studies indicate that CpG-ODN therapies can also be used for successful treatment of infections caused by bacteria, parasites or viruses. In these experiments, innate and adaptive immune responses against pathogens were augmented by CpG-ODN and subsequently induced resistance against infectious diseases. The stimulation of dendritic cells played a central role for the therapeutic effect of CpG-ODN. However, CpG-ODN can also have negative side effects, which accelerate disease progression in some viral infections. Clinical studies with CpG-ODN will determine their potential for the therapy of infectious diseases in humans.     

Synthesis and characterization of oligodeoxynucleotides containing 4-O-methylthymine

The carcinogenicity of N-nitroso compounds is believed to result from the alkylation of DNA, particularly on O-6 of the guanine and O-4 of the thymine residues. In order to study the base-pairing properties of 4-O-methylthymidine (T*) residues and the structural changes produced in DNA by the presence of this alkylated nucleoside, the oligodeoxyribonucleotides T*GCG, CGCAAGCTT*GCG, CGCGAGCTT*GCG, and CGCAAGCTTGCG were synthesized by the phosphotriester approach in solution. The 4-O-methylthymidine required for oligonucleotide synthesis was prepared by treating the 4-(3-nitro-1,2,4-triazolo) derivative of 3',5'-bis-O-(methoxyacetyl)thymidine with 1,8-diazabicyclo[5.4.0]-undec-7-ene (DBU) in methanol solution. The susceptibility of the 4-O-methyl group of T* toward nucleophiles enables this group of 4-O-methylthymidine-containing oligomers to be labeled by a direct exchange reaction with [13C]- or [14C]methanol in the presence of DBU. Although it has been previously suggested that 4-O-methylthymine forms stable base pairs with guanine, the thermal melting profiles of the double helices formed by these dodecamers suggest that the presence of 4-O-methylthymine paired to either adenine or guanine destablizes the helix. The melting curve of the sequence containing a 4-O-methylthymine residue base paired to guanine was biphasic and similar to that of an analogous sequence containing 6-O-methylguanine paired to thymine.     

Effects of N7-methylation, N7-platination, and C8-hydroxylation of guanine on H-bond formation with cytosine: platinum coordination strengthens the Watson-Crick pair

d ₆, applying concentration-dependent ¹H NMR spectroscopy: 9-ethylguanine, 7,9-dimethylguanine (7,9-DimeGH⁺), and 7,8-dihydro-8-oxo-9-methylguanine (8-O-9-MeGH), as well as three 9-ethylguanine complexes carrying different Pt(II) moieties at the N7 position. The association constants K for the Watson-Crick pairing schemes are by a factor 2–3 higher in the cases of platinated guanine complexes compared to the Watson-Crick pair between 9-ethylguanine and 1-methylcytosine (K=6.9±1.3 M⁻¹). Similar enhanced stabilities are observed for the pairs formed between 1-MeC and 7,9-DimeGH⁺ or 8-O-9-MeGH. The increase in N1H acidity of the guanine derivative upon modification at the N7 or C8 positions can be correlated with the association constants K; the result is a bell-shaped curve meaning that acidification initially stabilizes hydrogen bond formation up to a certain maximum; further acidification then leads to a destabilization. For two of the examples studied in solution, hydrogen bonding according to Watson-Crick between N7-platinated 9-ethylguanine and 1-methylcytosine has also been established by X-ray crystallography. Received: 29 December 1999 / Accepted: 15 February 2000     

Antisense oligodeoxynucleotides: synthesis, biophysical and biological evaluation of oligodeoxynucleotides containing modified pyrimidines.

6-Azathymidine, 6-aza-2'-deoxycytidine, 6-methyl-2'-deoxyuridine, and 5,6-dimethyl-2'-deoxyuridine nucleosides have been converted to phosphoramidite synthons and incorporated into oligodeoxynucleotides (ODNs). ODNs containing from 1 to 5 of these modified pyrimidines were compared with known 2'-deoxyuridine, 5-iodo-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, 5-fluoro-2'-deoxyuridine, 5-bromo-2'-deoxycytidine, and 5-methyl-2'-deoxycytidine nucleoside modifications. Stability in 10% heat inactivated fetal calf serum, binding affinities to RNA and DNA complements, and ability to support RNase H degradation of targeted RNA in DNA-RNA heteroduplexes were measured to determine structure-activity relationships. 6-Azathymidine capped ODNs show an enhanced stability in serum (7- to 12-fold increase over unmodified ODN) while maintaining hybridization properties similar to the unmodified ODNs. A 22-mer ODN having its eight thymine bases replaced by eight 6-azathymines or 5-bromouracils hybridized to a target RNA and did not inhibit RNase H mediated degradation.