Self-complementary Oligodeoxynucleotide Research Articles

5-Ethynylarylnaphthalimides as antitumor agents: Synthesis and biological evaluation

A set of 5-ethynylarylnaphthalimides was synthesized by Sonogashira cross-coupling reactions and evaluated for antiproliferative and antitopoisomerase II in vitro activities. Furthermore docking studies of these molecules as DNA-intercalators were carried out and the in vivo DNA-damaging activity was also determined with the model organism Saccharomyces cerevisiae. From the obtained results three naphthalimides 6, 13 and 14 showed strong topoisomerase II inhibitory activity. These three molecules also presented good docking scores as DNA-intercalators using a self-complementary oligodeoxynucleotide d(ATGCAT)2 as a model, and compounds 13 and 14 were among the most cytotoxic in the in vivo DNA-damaging activity.

Synthesis and Hybridization Properties of Modified Oligodeoxynucleotides Carrying Non‐Natural Bases

The impact of the presence of nonnatural bases on the properties of oligodeoxynucleotides has been studied. First, oligodeoxynucleotides carrying 2'-deoxyzebularine were prepared, and the stability of duplexes carrying this analogue was determined by DNA melting experiments. Melting temperatures and thermodynamic data indicated the preference of 2'-deoxyzebularine for 2'-deoxyguanosine, which behaves as a 2'-deoxycytidine analogue, forming a less stable base pair due to the absence of the amino group at position 4. Moreover, the duplex-hairpin equilibrium of a self-complementary oligodeoxynucleotide carrying several natural and nonnatural bases including 2'-deoxyzebularine as a central mispair, was studied. Depending on the base present in the middle of the sequence, it is possible to affect the stability of the bimolecular duplex modulating the duplex-hairpin equilibrium. Magnesium ions were shown to stabilize preferentially the bimolecular duplex form. The results indicate the importance of the modifications and the role of cations in shifting the structural equilibrium.

A Metal‐Coordinating DNA Hairpin Mimic

A self-complementary oligodeoxynucleotide containing a 6,6"-substituted terpyridine was found to adopt a highly stable, hairpin-like structure. In addition to serving as a hairpin-loop mimic, the terpyridine can act as a coordination site for metals. Thus, the binding of several divalent transition metals (Zn(2+), Co(2+), Ni(2+), Cu(2+) and Pd(2+)) to the terpyridine hairpin mimic was investigated. The terpyridine-modified hairpin mimic forms a stable secondary structure in the presence of these metals. The stability of the metal-coordinated hairpin mimic was found to be lower than in the absence of metal. Furthermore, the T(m) of the metallohairpin is strongly influenced by the type of the bound metal, with T(m)'s increasing in the order Co(2+) approximately Ni(2+) < Zn(2+) < Cu(2+) < Pd(2+). Model considerations suggest that a conformational change of the terpyridine ligand is required to allow coordination of the metal.

Solution structure of a DNA duplex containing mispair-aligned N4C-ethyl-N4C interstrand cross-linked cytosines.

The solution structure of an interstrand cross-linked self-complementary oligodeoxynucleotide containing directly opposed alkylated N(4)C-ethyl-N(4)C cytosine bases was determined by molecular dynamics calculations guided by NMR-derived restraints. The undecamer d(CGAAACTTTCG)(2), where C represents directly opposed alkylated N(4)C-ethyl-N(4)C cytosine bases, serves as model for the cytotoxic cross-links formed by bifunctional alkylating agents used in cancer therapy. The structure of the duplex shows the cross-link protruding into the major groove. An increase in the diameter of the DNA at the pseudoplatform formed by the cross-linked residues creates an A-DNA characteristic hole in the central portion of the DNA. This results in a centrally underwound base step and a number of subsequent overwinding steps leading to an overall axis bend toward the major groove. The structure shows narrowing of both minor and major grooves in the proximity of the cross-link. The perturbation leads to preferential intrastrand base stacking, disruption of adjacent canonical (A.T) base pairing, and buckling of base pairs, the extent of which diminishes with progression away from the lesion site. Overall, the distortion induced by the cross-link spreads over three base pairs on the 5'- and 3'-sides of the cross-link.

Structural studies of an oligodeoxynucleotide containing a trimethylene interstrand cross-link in a 5'-(CpG) motif: model of a malondialdehyde cross-link.

Malondialdehyde (MDA), a known mutagen and suspected carcinogen, is a product of lipid peroxidation and byproduct of eicosanoid biosynthesis. MDA can react with DNA to generate potentially mutagenic adducts on adenine, cytosine, and particularly guanine. In addition, repair-dependent frame shift mutations in a GCGCGC region of Salmonella typhimurium hisD3052 have been attributed to formation of interstrand cross-links (Mukai, F. H. and Goldstein, B. D. Science 1976, 191, 868--869). The cross-linked species is unstable and has never been characterized but has been postulated to be a bis-imino linkage between N(2) positions of guanines. An analogous linkage has now been investigated as a stable surrogate using the self-complementary oligodeoxynucleotide sequence 5'-d(AGGCG*CCT)(2,) in which G* represents guanines linked via a trimethylene chain between N(2) positions. The solution structure, obtained by NMR spectroscopy and molecular dynamics using a simulated annealing protocol, revealed the cross-link only minimally distorts duplex structure in the region of the cross-link. The tether is accommodated by partially unwinding the duplex at the lesion site to produce a bulge and tipping the guanine residues; the two guanines and the tether attain a nearly planar conformation. This distortion did not result in significant bending of the DNA, a result which was confirmed by gel electrophoresis studies of multimers of a 21-mer duplex containing the cross-link.

Studies of the mechanisms of adduction of 2'-deoxyadenosine with styrene oxide and polycyclic aromatic hydrocarbon dihydrodiol epoxides.

The mechanism of adduction of 2'-deoxyadenosine by styrene oxide and polycyclic aromatic hydrocarbon dihydrodiol epoxides has been explored using (15)N(6)-labeled adenine nucleosides. The extent of reaction at N1 versus N(6) was evaluated by (1)H NMR of the N(6) adducts after allowing Dimroth rearrangement to occur. Products arising from attack at N1 followed by Dimroth rearrangement exhibited a small two-bond (1)H-(15)N coupling constant (N1-H2 J approximately 13 Hz); products from direct attack exhibited a much larger one-bond (1)H-(15)N coupling constant (J approximately 90 Hz). In the case of styrene oxide, all of the N(6) beta adduct arose by initial attack at N1, whereas the majority (70-80%) of the N(6) alpha adducts came from direct attack. The styrene oxide reaction was also studied with a self-complementary oligodeoxynucleotide (24-mer) containing nine (15)N(6)-labeled adenine residues. NMR examination of the N(6) alpha- and beta-styrene oxide adducts isolated after enzymatic degradation of the 24-mer gave very similar results, indicating that N1 attack can occur readily even with a duplexed oligonucleotide. With the PAH dihydrodiol epoxides, only naphthalene dihydrodiol epoxide exhibited significant initial reaction at N1 (50%). No detectable rearranged product was seen in reactions with benzo[a]pyrene dihydrodiol epoxide or non-bay or bay region benz[a]anthracene dihydrodiol epoxide; interestingly, a small amount of N1 attack (5-7%) was seen in the case of benzo[c]phenanthrene dihydrodiol epoxide. It appears that initial attack at N1 is only a significant reaction pathway for epoxides attached to a single aromatic ring.

Non-Covalent Complexes between DNA-Binding Drugs and Double-Stranded Oligodeoxynucleotides: A Study by ESI Ion-Trap Mass Spectrometry

We developed an assay that utilizes electrospray ionization mass spectrometry (ESI-MS) to determine rapidly the noncovalent binding of drugs with oligodeoxynucleotides and to assess their relative affinities and stoichiometries. The method uses a set of self-complementary oligodeoxynucleotides that differ in length (6-mer to 12-mer), motif (GC-rich or AT-rich), and sequence, and these were annealed to form duplexes. To the oligodeoxynucleotides are bound a group of drugs (distamycin, Hoechst 33258, Hoechst 33342, berenil and actinomycin D abbreviated as D1, D2, D3, D4, and D5, respectively) that are classic minor-groove binders and intercalators. A second group (porphyrin H2TMpyP-4, metalloporphyrin CuTMpyP-4, FeTMpyP-4 and MnTMpyP-4 and [Ru(II)12S4dppz]Cl2 abbreviated as D6, D7, D8, D9, and D10) binds via mixed modes (i.e., groove binders and intercalators). The results confirm the binding stoichiometry and show preferred binding of minor-groove binders (distamycin, Hoechst 33258, Hoechst 33342, and berenil) to AT-rich oligomers and preferred interaction of the intercalator actinomycin D with GC-rich oligomers. The drugs H2TMpyP-4 and CuTMpyP-4 bind via mixed modes, whereas FeTMpyP-4 and MnTMpyP-4 interact by minor groove-binding only. Competitive binding experiments show that group-I drugs with duplex 5‘-CGCAAATTTGCG-3‘ have binding affinities in the order D3 > D2 > D1 > D4. The order for group-II drugs with duplex 5‘-ATATAT3-‘ is D6 ≈ D7 > D8 ≈ D9.

Inhibition of replication of fresh HIV type 1 patient isolates by a polypurine tract-specific self-complementary oligodeoxynucleotide.

A previously described self-complementary oligodeoxynucleotide termed triplex-forming oligodeoxynucleotide (TFO A), 54 bases in length, designed against the polypurine tract of HIV-1 RNA, inhibited viral replication at a 1 to 3 microM concentration in acutely infected cells, whereas antisense and scrambled sequence oligodeoxynucleotides were ineffective. Three HIV-1 viral isolates from patients of clinical categories A1, B, and C3 were transmitted to peripheral blood mononuclear cells and tested for production of p24 antigen and syncytium formation in the absence and in the presence of either TFO A or a control oligodeoxynucleotide of randomized sequence. No p24 antigen or syncytia were detected for up to 30 days when TFO A was added to the cells. Viability of the cells was found not to be affected by the drugs compared to controls within 2 weeks. Analysis of viral DNA synthesis by PCR for the LTR and gag gene indicated no DNA signal, suggesting that TFO A affects viral replication before formation of a DNA provirus. Measurements of the stability of TFO A indicate a half-life of about 2 hr. A two-dimensional computer fold analysis of TFO A suggested a self-complementary hairpin-loop configuration with GC-rich stems and single-stranded 5' and 3' ends. Since intracellular triplex formation may not be an efficient process, the observed inhibitory effect may be due to a direct inhibition of the RT and RNase H enzyme activities by the oligodeoxynucleotide. However, a triple-helix effect on the incoming RNA may play a role as well.

Selective abstraction of 2H from C-1' of the C residue in AGC.ICT by the radical center at C-2 of activated neocarzinostatin chromophore: structure of the drug/DNA complex responsible for bistranded lesion formation.

Glutathione-activated neocarzinostatin chromophore (NCS-Chrom) generates bistranded lesions at AGC.GCT sequences in DNA, consisting of an abasic site at the C residue and a strand break at the T residue on the complementary strand, due to hydrogen atom abstraction from C-1' and C-5', respectively. Earlier work showed that 2H from C-5' of T was selectively abstracted by the radical center at C-6 of activated NCS-Chrom, supporting a proposed model of the active-drug/DNA complex. However, since under the conditions used breaks at the T exceeded their inclusion in bistranded lesions, it was not clear what fraction of the hydrogen transfer represented bistranded lesions. Since virtually all abasic sites at the C are part of a bistranded lesions, hydrogen transfer from C-1' of C into the drug should reflect only the bistranded reaction. Accordingly, a self-complementary oligodeoxynucleotide 5'-GCAGCICTGC-3' was synthesized in which the C contained 2H at the C-1' position. In order to eliminate an 2H isotope effect on the transfer and to increase the extent of the bistranded reaction, an I residue was substituted for the G opposite the C residue. Sequencing gel electrophoretic analysis revealed that under one-hit kinetics, 37% of the damage reaction was associated with abasic site (alkali-labile break) formation at the C residue and 48% with direct strand breaks at the T residue. Thus, 74% of the damage involved a bistranded lesion. 1H NMR spectroscopic analysis of the reacted chromophore showed that 2H had been selectively transferred into the C-2 position to the extent of approximately 22%.(ABSTRACT TRUNCATED AT 250 WORDS)

Two-dimensional NMR studies of the conformational B → Z transition in the oligodeoxynucleotides d(CG)8and d(m5CG)8

The self-complementary oligodeoxynucleotide sequences d(CG)8 and d(m5CG)8 have been synthesized and characterized by COSY and NOESY experiments. In the respective B conformations, spectra are obtained with a resolution that is significantly higher than the one observed for the corresponding polydeoxynucleotides, d(CG) n . As a consequence, the signals from terminal bases can be resolved and identified for our systems. For the Z conformation, the lines are broader due to the higher viscosity of the solutions, and as a consequence of partial aggregation processes. The B → Z transition, induced by the addition of NaCl or MgCl2, respectively, can be monitored unambiguously in the NOESY spectra. In the B conformation, which is characterized by a 2′-endo sugar pucker and an anti-conformation of the bases with respect to the sugar ring, small interproton distances and hence strong NOE cross peaks are found for the proton pairs GH8/H2′ and CH6/H2′. In contrast, the bases are adopting a syn-conformation with respe...

Selective abstraction of 2H from C-5' of thymidylate in an oligodeoxynucleotide by the radical center at C-6 of the diradical species of neocarzinostatin: chemical evidence for the structure of the activated drug-DNA complex.

Use has been made of the mechanism of DNA deoxyribose damage by the ene-diyne-containing chromophore of the antitumor antibiotic neocarzinostatin to provide chemical evidence for the structure of the activated drug-DNA complex. Radical centers at C-2 and C-6 of the diradical form of the glutathione-activated chromophore abstract hydrogen atoms from C-1' of the C residue and C-5' of the T residue in AGC.GCT to generate a bistranded lesion consisting of an abasic site at C and a strand break at T. This laboratory has proposed a molecular model for the drug-DNA interaction in which the naphthoate moiety of the chromophore intercalates between A.T and G.C, placing the diradical core in the minor groove, so that the radical centers at C-6 and C-2 are close to C-5' of T and C-1' of C, respectively. To determine which radical center abstracts one of the hydrogen atoms from C-5', the self-complementary oligodeoxynucleotide GCAGCGCTGC was synthesized with 2H at both 5' positions of the T residue and treated with glutathione-activated chromophore. Sequencing-gel electrophoresis showed that drug attack was limited to the T and C residues and that abstraction of 2H from C-5' exhibited a small isotope selection effect of 1.25. 1H NMR spectroscopic examination of the reacted chromophore, isolated by HPLC, indicated that 2H was selectively abstracted by C-6, providing experimental corroboration of the model and further elucidating the chemical mechanism. Since direct strand breakage at the T residue exceeds (44% more) abasic site formation at the C residue, other models of drug-DNA interaction leading to only single-strand breaks are also considered.

The use of Raman spectroscopy to characterize double B/Z conformational junctions in DNA

Raman spectra of the duplex of the self-complementary oligodeoxynucleotide, d[TTCGCGCGCGAA] at various salt concentrations and temperatures have been acquired. Two methods are described which indicate that the intensities of the bands at 680 cm−1 and 625 cm−1 can be analyzed using the band assignments of Tsuboi, Nishimura and colleagues to determine the relative number of CG base pairs in the Z form. The absence of bands characteristic of AT base pairs in the Z form rules out the occurrence of the Z form in the AT portion of the duplex. From these measurements the structure of the double helical duplex in concentrated salt solutions can be described as having four of the six interior CG base pairs in Z form flanked by two CG base pairs in the junction and the two AT base pairs in a short B-form segment.

The left-handed Z-DNA conformation in oligodeoxynucleotides containing different amounts of AT base pairs: a far UV circular dichroism study.

A number of fully self-complementary oligodeoxynucleotides have been synthesized and examined for their ability to assume the left-handed Z-DNA conformation in high salt solutions. The B- and Z-forms are identified by circular dichroism spectra, covering both the long- (220-300 nm) and short-wavelength (185-220 nm) regions, the latter showing CD bands very useful for identifying the sense of the helix winding. The main results of the study can be summarized as follows: a) sequences composed by AT and CG blocks do support the B to Z transition, even when the AT contents amounts to 50%; b) the occurrence of consecutive purine-purine or pyrimidine-pyrimidine dyads does not inhibit the B to Z transition, although a stronger reduction of water activity is required; c) (AC)n and (GT)n containing oligonucleotides do undergo the B to Z transition in solution; d) a millimolar quantity of Ni2+ concomitant with 5 M NaClO4 is found to be very effective in bringing about the B to Z transition in most of the sequences considered in this study.

Repair of O6-methylguanine, O6-ethylguanine, O6-isopropylguanine and O4-methylthymine in synthetic oligodeoxynucleotides by Escherichia coli ada gene O6-alkylguanine-DNA-alkyltransferase.

Self-complementary oligodeoxynucleotides have been synthesized containing O6-methylguanine (O6meG), O6-ethylguanine (O6etG), O6-isopropylguanine (O6iprG) and O4-methylthymine (O4meT). They anneal in solution to give double-stranded DNA. These double helices have been used as substrates for the DNA repair protein O6-alkylguanine-DNA-alkyltransferase coded for by the ada gene of Escherichia coli. The repair followed second-order chemical kinetics. O6meG was repaired by the 19-kd transferase at a rate of 2.54 x 10(7) M-1 s-1 which is close to the theoretical limit for a diffusion-controlled reaction; O6etG and O4meT are repaired 1,000 and 10,000 times more slowly. The 39-kd alkyltransferase (which is precursor to the 19-kd form) and the 19-kd transferase repaired O6etG at similar rates. O6iprG was not repaired. The repair of oligomers containing O6meG was only slightly inhibited by the presence of nonalkylated oligomers. Oligomers containing O6etG were only slightly more effective as inhibitors of repair than the nonalkylated oligomers, indicating that the transferase does not bind selectively to alkylated DNA. Parallel structural studies have shown that O6-alkylguanine:C and O4-alkylthymine:A base pairs have a similar geometry with the alkylated base displaced into the major groove of the DNA in contrast to O6-alkylguanine:T and O4-alkylthymine:G base pairs which retain the Watson-Crick alignment with N1 of the purine juxtaposed to N3 of the pyrimidine. Measurement of the rate of repair of these different base pairs suggests that pairs with the alkyl group exposed in the major groove may be repaired more rapidly than those with the alkyl group more deeply buried in the helix.

The synthesis of 2-pyrimidinone nucleosides and their incorporation into oligodeoxynucleotides.

The synthesis of 1-(beta-D-2'-deoxyribosyl)-2-pyrimidinone (dK) and its 5-methyl derivative (d5) from 2'-deoxycytidine or 2'-deoxythymidine, respectively, via silver oxide oxidation of 4-hydrazinopyrimidines is described. The necessary hydrazine substituted pyrimidine nucleosides have been prepared by transamination of a protected cytidine derivative or by addition/elimination reactions to an O4-sulfonated thymidine derivative. Oxidation of the 4-hydrazino pyrimidines was complicated by a competing hydrolytic reaction which generated 2'-deoxyuridine or 2'-deoxythymidine. However, in the presence of an organic base such as triethylamine, oxidation became the predominant reaction. After suitable protection and formation of the 3'-phosphoramidite derivatives, these modified nucleosides were incorporated into seven self-complementary oligodeoxynucleotides by chemical synthesis using phosphite triester methodology. Oligodeoxynucleotides were prepared such that dA-dT and dG-dC base pairs were substituted by dA-d5 or dG-dK base pairs, respectively. Both circular dichroism spectra and thermal denaturation studies were used to characterize the modified oligodeoxynucleotides.

Study of structure, base-pair opening kinetics and proton exchange mechanism of the d-(AATTGCAATT) self-complementary oligodeoxynucleotide in solution.

Using proton magnetic resonance, we have investigated the structure and the base-pair opening kinetics of the d-(AATTGCAATT) self-complementary duplex. All the non-exchangeable (except H5',5") and most exchangeable proton resonances have been assigned. The structure belongs to the B family. Imino proton exchange, measured by line broadening, longitudinal relaxation and magnetization transfer from water, is catalyzed by proton acceptors. The base-pair lifetimes, obtained by extrapolation of the exchange times to infinite concentration of ammonia are 2 and 3 milliseconds for internal A.Ts and 18 ms for G.C at 15 degrees C. In the absence of added catalysts, the imino proton of the first A.T base pair exchanges faster than that of the unpaired thymidine of the duplex formed by the sequence d-(AATTGCAATTT). This gives strong evidence for intrinsic exchange catalysis. The exchange of adenine amino protons from the closed state has been observed. Hence amino proton exchange is ill-suited for the investigation of base-pair opening kinetics.

Conformational perturbation due to an extra adenosine in a self-complementary oligodeoxynucleotide duplex.

AbstractThe conformation of an oligodeoxynucleotide pentadecamer, d(CGCGAAATTTACGCG), self‐complementary except for an additional adenosine nucleotide at position 11, has been investigated with nmr spectroscopy. This oligomer was found to exist predominantly in the duplex form in solution rather than in the hairpin loop form, as observed previously for an analogous 13‐mer sequence. Nuclear Overhauser effects indicate that the B‐form conformation is disrupted by the extra A base, which forms a wedge within the duplex, producing a bent junction and an overall S‐like structure. A downfield‐shifted phosphate resonance was assigned using a two‐dimensional 1H–31P correlation method, and was found to be P12‐13. This and other results indicate that the extra A causes conformational distortions at some distance along the duplex structure.

Cross-linking of bromodeoxyuridine-substituted oligonucleotides to the EcoRI and EcoRV restriction endonucleases.

We have synthesized several self-complementary oligodeoxynucleotides which contain bromodeoxyuridine in various positions within and outside of the recognition sequence for the EcoRI and EcoRV restriction endonucleases. These oligodeoxynucleotides are cleaved in the presence of Mg2+ by their respective enzyme. Upon irradiation by long-wavelength ultraviolet light and in the absence of Mg2+ they are cross-linked in low yield to their enzymes, forming 1:1 and 1:2 (oligodeoxynucleotide:enzyme subunit) adducts. Cross-linking occurs with both specific and non-specific complexes. With EcoRI the site of cross-linking was determined to be at or close to Met-137, i.e. in a region of the molecule implicated by other studies from our laboratory [Scholtissek et al. (1986) J. Biol. Chem. 261, 2228-2234] in the binding and cleavage of the substrate.