1H and 31P nuclear magnetic resonance studies of the differences in DNA deformation induced by anti-tumoral 7H-pyrido[4,3-c]carbazole dimers.

Ditercalinium (2,2'-[( 4,4'-bipiperidine]-1,1'-diyldi-2,1-ethane-diyl) bis-[10-methoxy-7H pyrido[4,3-c]carbazolium)tetramethane sulfonate (NSC 366241], a DNA bis-intercalating compound, is a potent anti-tumoral rigid dimer. Previous studies have shown that a reduced flexibility of the linking chain of such a dimer is essential for its biological activity. In order to understand, at the molecular level, the mechanism of action and the structure-activity relationships of this series of DNA intercalators, new dimers with additional methylene groups between the two piperidine rings have been synthesized. Addition of one methylene group in the chain preserved the activity, whereas addition of two methylene groups reduced the cytotoxicity, which finally disappeared when three methylene groups were inserted. Therefore, the study of the interaction of dimers bearing no (202), two (222) and three (232) methylene groups with the self-complementary hexanucleotide d(CGATCG)2 have been investigated by 1H and 31P nuclear magnetic resonance studies. The results reported here indicate that all dimers bis-intercalate into the minihelix. The intermolecular nuclear Overhauser effects (NOEs) between the dimers and the nucleotide lead to the conclusion that the three dimers intercalate with their rigid bis-ethyl bipiperidine chain fitting the major groove of the helix. Inter-residue nuclear Overhauser effects at the DNA level, as well as induced shifts, are discussed in relation to the conformational changes induced in DNA upon intercalation and to the different activity of the dimers.

Molecular structure of a complete turn of A-DNA.

We have determined the crystal structure of the dodecamer d(CCCCCGCGGGGG), showing for the first time a complete turn of A-DNA. It has average structural parameters similar to those determined in fibres. Nevertheless it shows a considerable local variation in structure which is in part associated with the presence of a bound spermine molecule. We conclude that the local DNA conformation does not only depend on the base sequence, but may be strongly modified upon interaction with other molecules. In particular, the CpG sequence, which is found in hypersensitive regions of the genome, appears to be able to easily change its conformation under external influences.

500 MHz 1H-NMR study of the interaction of daunomycin with B and Z helices of d(CGm5CGCG).

The interaction of daunomycin with B and Z helices of a self-complementary DNA fragment d(CGm5CGCG) in solution was studied by 1H-NMR spectroscopy at 500 MHz. The results show that the B-Z transition kinetics is not affected by addition of daunomycin. Daunomycin binds exclusively to the B form of d(CGm5CGCG). Z exchanges with B while the latter also exchanges with the B duplex-daunomycin complexes.

Simple chemical synthesis and hybridization properties of non-radioactive DNA probes.

A simple chemical method for the synthesis of non-radioactive DNA probes is described: triazolyl-containing sequences were built by incorporation of 4-triazolylpyrimidin-2-ones instead of cytidines during oligodeoxyribonucleotide synthesis. The activating triazolyl groups were then displaced by a diamine which was further derivatized by a label, such as biotin. Synthesized DNA probes were oligonucleotides complementary to a cloned human antithrombin III DNA sequence. These probes, containing the same label at different positions of the sequence, were hybridized to their target DNA immobilized on nitrocellulose. Their hybridization specificity and stability were studied. Hybrid detection was performed either colorimetrically by the streptavidin-alkaline phosphatase-based system or by autoradiography after 5'-32P labeling of the probes: 15 fmol (0.05 microgram) of complementary sequence could be visualized in the two cases.

1H NMR study of the structure of a pyridocarbazole dimer-d[CpGpCpG] complex.

The structure of the complexes formed between a 7H-pyridocarbazole dimer (ditercalinium) or the corresponding monomer and d[CpGpCpG] is analyzed in aqueous solution by 270 MHz 1H NMR. In both cases the strong upfield shifts observed on most aromatic resonances are assigned to the formation of intercalated complexes. Bisintercalation of the dimer in the tetranucleotide minihelix is then observed at pH 5.5. The observation of intermolecular negative NOEs induced to some drug resonances by irradiation of sugar protons confirms these conclusions. The orientation of the ligand in the intercalation site is discussed.

2-l-Rhamnopyranosyl[1,2,4]triazolo[1,5-a]pyridine. 4' and 3' Oxidation products. Synthesis and structure-activity relationships.

A series of 2-alpha-L-rhamnopyranosylnitro[1,2,4]triazolo[1,5-a] pyridine C-nucleosides was synthesized from the condensation oa thioiminoether with nitro-2-pyridylhydrazines. Catalytic reduction afforded the corresponding amino derivative. A 1',2' unsaturated C-nucleoside was also obtained by two different routes. Selective oxidation gave the 3'- and 4'-ketonucleosides. The cytotoxic properties of the nucleosides, as well as their effect on viral transformation and replication, were described. The nitro derivatives inhibit viral replication, but at toxic doses; the introduction of a keto function leads to a product which inhibits the replication of murine leukemia virus (MuLV) at noncytotoxic concentrations. The amino derivatives have no significant antiviral effect.

A general procedure for assigning the 31P spectra of drug-oligonucleotide complexes.

Taking advantage of the slow exchange at the NMR time scale occurring in drug oligonucleotides complexes the 31P signals in the bound forms are assigned by using 31P NMR two dimensional chemical exchange. This technique was applied to complexes between Actinomycin D and d[CpGpCpG] or d[m5CpGpm5CpG]. As compared to the labelled 17O, 18O this method proved to be a powerful and unique way to assign 31P in broad spectrum or with long oligonucleotides.

NMR studies of tris-intercalation: solution structure and interaction of d(CTTCGCGCGAAG) with an acridine trimer.

Tris-intercalation of an acridine trimer into the self-complementary dodecanucleotide d(CTTCGCGCGAAG) has been studied, in solution, by means of 1H and 31P nuclear magnetic resonance. In a first step all the non-exchangeable protons (except H5', H5"), the imino protons and seven of the eleven phosphorus have been assigned. The dodecanucleotide is shown to adopt a double helical B-type structure. Most of the sugar puckers are in the O1'endo range, those of the internal guanosines being closer to C2'endo. Deviations from the canonical B structure are observed in the base stacking and the phosphodiester torsional angles at the 3T4C5G stretch. The addition of an acridine trimer to the base-paired dodecanucleotide leads to the conclusion that the trimer, which is in slow exchange at the NMR time scale, tris-intercalates into the three C(3'-5')G sites of the central core, according to the excluded site model. This is evidenced by the large (1.4 ppm) upfield shift experienced by the imino protons of the three internal guanines and the shielding undergone by the acridine ring protons. Tris-intercalation is also supported by the downfield shift experienced by 6 out of the 22 phosphorus. Two of them are shifted by nearly 2 ppm, a shift range reported for oligonucleotides complexed to actinomycin D; this suggests that the structure of the backbone of the dodecanucleotide is altered.

IR and UV studies on stability and conformations of short DNA duplexes containing a no-base residue: coexistence of B and Z conformations.

Tridecamers containing a central no-base residue (X) have been synthesized and hybridized to their complementary strands, so as to constitute duplexes consisting of two hexamers separated by central mismatched X-A or X-T pairs. The effect of the introduction of this deoxyribose derivative on duplex stability was investigated by measuring UV absorbance as a function of salt concentration and temperature. As expected, the duplexes containing the abnormal base pairs (X-T and X-A) are less stable when compared to the totally complementary duplexes (A-T and T-A). The X-T mismatched duplex shows the most unstable thermodynamical behaviour. The conformational changes of these duplexes were studied by IR spectroscopy in condensed phase as a function of water content. At high relative humidity, the IR spectra show that these tridecamers form B-type double stranded duplex structures. If the water content is decreased, only the duplexes m5CGm5CGCTXAGCTTC GCGCGAATCGAAG and, to a lesser degree, m5CGm5CGCTXAGCTTC GCGGCATTCGAAG undergo a partial B---Z transition involving the methylated hexamer, the conformation of the second segment remaining of the B type. These results show that only one apurinic residue leads to a flexible junction between B and Z forms in a short duplex containing 5-methyl-2'-deoxycytidines.

Reassessment of structural characteristics of the d(CGCG)2:actinomycin D complex from complete 1H and 31P NMR.

Complexes formed between Actinomycin D (ActD) and the tetranucleotides d(AGCT)2 and d(CGCG)2 were studied in detail by one and two-dimensional 1H and 31P NMR. The 31P two dimensional chemical exchange experiment, at room temperature on saturated complexes (1:1), showed unambiguously that the asymmetrical phenoxazone ring binds to the unique GC site under the two possible orientations in the d(AGCT)2 tetranucleotide but adopts a single orientation in the d(CGCG)2 tetranucleotide. For the d(CGCG)2:Act D saturated complex, complete assignments of all protons and phosphorus signals of the two-nucleotide strands, as well as of the two cyclic pentapeptide chains has allowed us to study in details the conformational features of the complex from NOE and coupling constants analysis. The tetranucleotide remains in a right-handed duplex, but the sugar puckers are modified for residues at the intercalation site. A uniform C2' endo pucker is observed for residues on the strand facing the quinoid side of the phenoxazone ring while a C2' endo-C3-endo equilibrium about 60% of C2' endo is proposed for the two residues on the strand facing the benzenoid side of the phenoxazone ring. In contrast to previous studies on ActD-DNA interactions, we have been able to measure the 3J phosphorus-proton coupling constants at the intercalation site but also adjacent to it, showing that 31P chemical shifts are not simply related to the backbone conformation. Molecular mechanics calculations, using empirical distances deduced from NOE effects as restrained distances during minimizations, led to a model differing mainly from those previously published by orientation of the N methyl groups of both N-Methyl-Valines.

Infrared spectral studies of the non regularly alternating purine-pyrimidine hexamers d(m5CGGCM5CG), d(CBr8GGCCBr8G) and d(CGCGGC).

The oligonucleotides d(m5CGGCm5CG), d(CBr8GGCCBr8G) and d(CGCGGC) have been prepared and studied by infrared spectroscopy. The three sequences contain two GC pairs which are out of purine-pyrimidine alternation with the rest of the sequence. From the IR data of the d(m5CGGCm5CG) hexamer, it is shown that all of the dG residues adopt a syn conformation. The marker IR bands for the C3' endo syn conformation are at 1410, 1354, 1320 and 925 cm-1 whereas those for the C2' endo anti conformation at 1420, 1374 and 890 cm-1 are clearly absent. This result implies that the two adjacent guanines of the d(m5CGGCm5CG) sequence are in syn conformation. It is suggested that duplex formation occurs in d(CGCGGC) films and that all of the guanines are in syn conformation. In contrast, the central non-brominated guanine of the d(CBr8GGCCBr8G) hexamer is found in anti conformation, as expected in a Z type structure of the non-alternating region.

B,Z conformations and mechanism of the Z-B-coil transitions of the self-complementary deoxy-hexanucleotide d(C-G-m5C-G-C-G) by 1H-NMR and CD spectroscopy.

The helical structures of d(C-G-m5C-G-C-G) were studied in aqueous solution at various salt concentrations and temperatures by CD and 1H-NMR spectroscopy. At room temperature only the B form is observed in 0.1 M NaCl whereas the B and Z forms are simultaneously present in 1.8 M NaCl. At high salt concentration (4 M NaCl) the Z form is largely predominant (greater than 95%). The Z form proton resonances were assigned by using the polarisation transfer method (between B and Z at 1.8 M NaCl) and by proton-proton decoupling (at high salt concentration). The Z-B-Coil transitions were studied as a function of temperature with the 1.8 M NaCl solution. At high temperature (95 degrees C) only the coil form (S) is present. Below 55 degrees C the coil proportion is negligible, and the B-Z exchange is slow. The disappearance of the coil gives rise at first to the B form and on lowering the temperature the Z proportion increases to the detriment of the B form. Proton linewidth, relaxation and polarisation transfer studies confirm the conclusion in the previous report on d(m5C-G-C-G-m5C-G) (Tran-Dinh et al Biochemistry 1984 in the press) that Z exchanges only with B whereas the latter also exchanges with S,Z in equilibrium B in equilibrium S. The present data show that even at high salt concentration where only the Z form of d(C-G-m5C-G-C-G) is observed the Z-S transition also passes through the B form as an intermediate stage. The B-Z transition takes place when the Watson-Crick hydrogen bonds are firmly maintained and is greatly favoured when there are three hydrogen bonds between the base-pairs.

Z helix-coil transition of d(C-Br8G-C-G-C-Br8G) studied by CD, 1H-NMR and IR spectroscopies.

The conformation of d(C-Br8G-C-G-C-Br8G) in aqueous solution was studied by CD and 1H-NMR spectroscopy and in condensed phase by IR spectroscopy. Whether in 0.1 M or 3 M NaCl solution or in film the only double helical structure adopted by brominated d(C-G)3 oligomer is the Z form. The IR spectrum of the film presents all the characteristic absorptions of the Z conformation and in particular is indicative of a syn conformation for the central guanosine as well as for the brominated one. Imino proton resonances of d(C-Br8G-C-G-C-Br8G) demonstrating the duplex formation were observed up to 60 degrees C. It is interesting to note that the significant highfield shifts of the dC H5" exocyclic sugar protons characteristic of the non exchangeable proton spectra of d(C-G)3 containing 5-methyl dC residues in the Z form were also detected in the proton spectrum of brominated oligomer. Whereas formation of the Z helix of methylated d(C-G)3 oligomers dependent on the salt concentration was found to occur via the preliminary formation of a B helix even in 4 M NaCl solution, the Z helix of d(C-Br8G-C-G-C-Br8G) is obtained directly from the coil form. However, IR data suggest that in the Z form of d(C-Br8G-C-G-C-Br8G), the overlapping of the base planes should be slightly different in comparison with the stacking observed in d(C-G)3 crystals. The kinetic data (activation energy and lifetime) of the Z helix-coil transition of brominated d(C-G)3 are compared to those of the B helix-coil transition observed for methylated d(C-G)3 in 0.1 M NaCl solution while the thermodynamic data of these two reactions (enthalpy and midpoint temperature) are slightly different.

Simple preparation of 1,2-dipalmitoyl-sn-glycero-3-phosphoric acid and deuterated choline derivatives.

Analytically pure 1,2-dipalmitoyl-sn-glycero-3-phosphoric acid was prepared in gram amounts, using a simplified version of a previous procedure. The main step, enzymatic cleavage of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine with freshly extracted phospholipase D, was performed in the presence of chloroform and the crude phosphatidic acid was purified by silica gel column chromatography. The interest of the method was illustrated by the synthesis of two dipalmitoyl phosphatidylcholines selectively deuterated on the polar headgroup.

Nuclear magnetic resonance study of d-TGGCCA in solution.

The 1H NMR spectrum of a self complementary deoxyhexanucleotide (d-TGGCCA) in water solution has been completely and unambiguously assigned by use of two-dimensional techniques and phosphorus off-resonance decoupling experiments. The helix-coil transition has been monitored by following most of the non-exchangeable protons against temperature and was shown to be a cooperative dissociation-association process. Sequential fraying is sizable even at low temperature. Considerations of coupling constants and of Nuclear Overhauser effects show the helix to be of the B-DNA type.

Conformational studies of d(m5CpGpm5CpG) and d(CpGpCpG) by 1H and 31P NMR.

Exhaustive conformational studies of d(CpG)2 and d(m5CpG)2, two convenient targets for DNA bisintercalating drugs, have been carried out by 1H and 31P NMR in low salt concentration and in the presence of 30% ethanol. Unambiguous 31P assignments of the B for are obtained with low-power heteronuclear decoupling experiments, while 31P assignments in the Z form are obtained by two-dimensional homonuclear chemical exchange experiments. The 31P chemical shifts and 3JH3'P coupling constants studied at various temperatures in methylated and non-methylated tetranucleotides, are interpreted as resulting from conformational differences between the compounds. These features are corroborated by homonuclear proton nuclear Overhauser effect experiments showing the steric role of the 5-methylcytosine in the induction of an alternating B form in d(m5CpG)2.

Modified oligonucleotides as alternatives to the synthesis of mixed probes for the screening of cDNA libraries.

Two simple alternatives to the synthesis of mixed oligodeoxyribonucleotide probes are described: mixed deblocking of triazolodeoxynucleosides for the A/G and C/T degeneracies or incorporation of 2-amino-2'-deoxyadenosines into a determined sequence for a higher stability of the hybridization duplexes. The synthetic oligodeoxyribonucleotides obtained were successfully tested in comparison with a classical mixed probe for their capacity to identify specific cDNA clones of human antithrombin III. The results are discussed with respect to the utilization of these synthetic oligonucleotides as hybridization probes for the isolation of cloned cDNA sequences.

Processing of complex heteroduplexes in Escherichia coli and Cos-1 monkey cells.

Upon transformation into Escherichia coli or Cos-1 monkey cells, heteroduplex DNA made of two sequences containing many nucleotide mismatches yields a wide array of different molecules, some with a patchwork structure. Thus, complex heteroduplexes can be processed to generate many genetic variants.

[Study of helix-coil transition of an oligodeoxyribonucleotide with self-complementary sequence d-ApCpApTpGpT by 1H-NMR]. / Etude de la transition hélice-pelote d'un oligodéoxyribonucléotide à séquence autocomplémentaire d-ApCpApTpGpT par RMN-1H.

The helix-coil transition of dACATGT was investigated by 1H-NMR spectroscopy at 276 and 400 MHz. The results suggest that the fraying process only consists of two stages: The AT extreme base pairs open at first then the GC internal and AT central base pairs open simultaneously at higher temperatures. The midpoint temperature of each base pair, the helix-coil dissociation constants and the corresponding enthalpy were determined from the delta = f (t degree) curves of non exchangeable protons. The unusual line width of the H1, proton resonance of the cytidine residue, observed at t less than 42 degree C was attributed to the fact that this proton is located in the proximity of the (cytoxine) C = O ... H2N (guanine) hydrogen bond.