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PubMed1998

RNase H1 can catalyze RNA/DNA hybrid formation and cleavage with stable hairpin or duplex DNA oligomers.

Cleavage of a RNA target site by RNase H1 from Escherichia coli was examined in the presence of complementary DNA sequences in the form of single-stranded, duplex, and hairpin structures. The target site was a 15 nt sequence in the middle of a 79 nt RNA transcript. DNA molecules employed included seven single-stranded oligodeoxynucleotides 10 or 15 nt long, and five hairpin DNAs each with a 10 bp stem and 5 nt loop. The loop and 3' side of the stem of two of the hairpin DNAs were fully complementary to the target site, while the other hairpin DNAs had sequence changes. A 10 bp duplex DNA with one strand complementary to the target site was also employed. A gel electrophoresis mobility shift assay examined hybrid formation between the RNA and the single-stranded 15 nt DNA and two hairpin DNAs that contained 15 complementary bases. RNA titration of the 32P-labeled single-stranded DNA produced a shifted band indicative of RNA/DNA complex formation. No RNA/DNA complex was detected when the more stable (Tm = 71 degrees C) hairpin DNA was combined with excess RNA. The less stable hairpin DNA (Tm = 62 degrees C) showed a small amount ( approximately 8%) of hybrid formation. Thermodynamic analysis of RNA binding to the DNAs was in qualitative agreement with the results. Although no RNA/DNA hybrid was expected from thermodynamic calculations, a RNase H assay at 25 degrees C showed that hairpin or duplex DNAs with a 10 nt complementary sequence catalyzed RNA degradation. A complementary loop sequence in the hairpin DNA was not required. Cleavage of the RNA did not occur with hairpin DNAs containing three or four noncomplementary bases in the stem. The results show that RNase H can promote the formation and cleavage of a RNA/DNA hybrid between an RNA site and a base paired strand of a stable hairpin or duplex DNA at temperatures below their Tm.

Bacterial Proteins↗

PubMed1996

Conformational properties of Z-forming DNA oligomers bearing terminal unpaired bases.

Three sets of semi-self-complementary deoxyribonucleotide decamers with the sequence XX-(5meCG)4, (5meCG)4-XX, or Y-(5meCG)4-Y, where XX = AA, CC, GG, or TT and Y = A, C, G, or T, were synthesized along with the self-complementary octamer (5meCG)4. The 8-mer duplex readily undergoes a B-to-Z conformational conversion upon increasing the NaCl concentration with a transitional midpoint of approximately 1.1 M NaCl. The 10-mers should form 8-bp duplexes a with core sequence of [(5meCG)4]2 with 5'-XX overhangs, 3'-XX overhangs, or 5',3'-Y/Y mismatches. Circular dichroism was employed to determine the conformations of all oligomers. Salt titrations were performed to measure the effect of overhangs and terminal mismatches on the B-to-Z conversion. In general, the presence of 5'-XX overhangs results in a transition midpoint equal to or slightly higher than the control, whereas the presence of 3'-XX overhangs results in a transition midpoint slightly lower than the control. The 3'-CC and 5'-GG overhangs are exceptions, with transition midpoints much higher than the control. These oligomers apparently form duplexes with 5',3'-C/C or 5',3'-G/G mismatches abutting a [(G5meC)4]2 duplex core. The presence of terminal mismatches in the third set of oligomers results in transition midpoints higher than the control. Ultraviolet absorbance methods were used to evaluate the effect of the various stacking motifs of the 10-mers on the thermodynamics of melting relative to the 8-mer for both B and Z conformations. We found that in both the B and Z conformations, the presence of an overhang stabilizes the [(5meCG)4]2 duplex, with the 5' overhangs having a greater stabilizing effect relative to the 3' overhangs. The presence of 5',3'-Y/Y mismatches also imparts a stabilizing effect on the control 8-mer in both the B and Z conformations. These results are discussed in terms of stacking interactions of the terminal unpaired bases.

Base Composition↗

PubMed2001

2.4 A crystal structure of an oxaliplatin 1,2-d(GpG) intrastrand cross-link in a DNA dodecamer duplex.

(1R,2R-Diaminocyclohexane)oxalatoplatinum(II) (oxaliplatin) is a third-generation platinum anticancer compound that produces the same type of inter- and intrastrand DNA cross-links as cisplatin. In combination with 5-fluorouracil, oxaliplatin has been recently approved in Europe, Asia, and Latin America for the treatment of metastatic colorectal cancer. We present here the crystal structure of an oxaliplatin adduct of a DNA dodecanucleotide duplex having the same sequence as that previously reported for cisplatin (Takahara, P. M.; Rosenzweig, A. C.; Frederick, C. A.; Lippard, S. J. Nature 1995, 377, 649-652). Pt-MAD data were used to solve this first X-ray structure of a platinated DNA duplex derived from an active platinum anticancer drug other than cisplatin. The overall geometry and crystal packing of the complex, refined to 2.4 A resolution, are similar to those of the cisplatin structure, despite the fact that the two molecules crystallize in different space groups. The platinum atom of the [Pt(R,R-DACH)](2+) moiety forms a 1,2-intrastrand cross-link between two adjacent guanosine residues in the sequence 5'-d(CCTCTGGTCTCC), bending the double helix by approximately 30 degrees toward the major groove. Both end-to-end and end-to-groove packing interactions occur in the crystal lattice. The latter is positioned in the minor groove opposite the platinum cross-link. A novel feature of the present structure is the presence of a hydrogen bond between the pseudoequatorial NH hydrogen atom of the (R,R)-DACH ligand and the O6 atom of the 3'-G of the platinated d(GpG) lesion. This finding provides structural evidence for the importance of chirality in mediating the interaction between oxaliplatin and duplex DNA, calibrating previously published models used to explain the reactivity of enantiomerically pure vicinal diamine platinum complexes with DNA in solution. It also provides a new kind of chiral recognition between an enantiomerically pure metal complex and the DNA double helix.

Antineoplastic Agents↗

PubMed1984

Nucleotide sequence preference at rat liver and wheat germ type 1 DNA topoisomerase breakage sites in duplex SV40 DNA.

The site specificities of the type 1 DNA topoisomerases (topo 1) from rat liver and wheat germ were investigated. The nucleotide sequence at break sites on duplex SV40 DNA were determined for 245 wheat germ topo 1 sites and 223 rat liver topo 1 sites over a region of 1781 nucleotides. The enzymes from the two different sources show similar, but not identical patterns of DNA strand breakage. The sites occur frequently, but are not broken with equal probabilities. Major sites of breakage occur on the average every one to two turns of the helix, thus if sites of breakage accurately represent topo 1 sites of activity, the DNA sequence alone would appear to place few limits on the access of the enzyme to DNA. Sequences around the strongest sites for both enzymes show a bias in base composition for the four nucleotides immediately 5' to the break site (-4 to -1 positions), but no bias is observed 3' to the site of breakage. Consensus sequences for both enzymes were determined. Variations from the consensus sequence appear to affect the two enzymes differently and may account for the differences observed in the specificity of breakage.

Animals↗

PubMed1991

End-filling of an oligonucleotide duplex containing an MDBP site in the human HSP70 promoter inhibits protein-DNA complex formation.

A site from the promoter region of the human hsp70 gene binds with a high affinity to the ubiquitous mammalian protein called methylated DNA-binding protein (MDBP) when it is present in a CpG-methylated oligonucleotide duplex with only 14 base-pairs. Binding to this site is dependent upon CpG methylation. Surprisingly, when the same methylated sequence is present in a duplex that has 22 or more base-pairs, binding to this protein is greatly inhibited. Such a requirement for a short duplex region is seen only in certain of the cytosine methylation-dependent binding sites for this protein and is proposed to reflect differences in the conformation of the duplex due to small differences in the nucleotide sequence.

Base Sequence↗

PubMed1996

Observation of the A-DNA to B-DNA transition during unrestrained molecular dynamics in aqueous solution.

A large challenge in molecular dynamics (MD) simulations of proteins and nucleic acids is to find the correct "experimental" geometry when a simulation is started a significant distance away from it. In this study, we have carried out four unrestrained approximately 1 ns length MD trajectories in aqueous solution on the DNA duplex d(CCAACGTTGG)2, two beginning in a canonical A-DNA structure and two beginning in a canonical B-DNA structure. As judged by root-mean-squared coordinate deviations, average structures computed from all four of the trajectories converge to within approximately 0.8 to 1.6 angstroms (all atoms) of each other, which is 1.3 to 1.7 angstroms (all atoms of the central six residues from each strand) and 3.1 to 3.6 angstroms (all atoms) away from the B-DNA-like X-ray structure reported for this sequence. To our knowledge, this is the first example of multiple nanosecond molecular dynamics trajectories with full representation of DNA charges, solvent and long range electrostatics that demonstrate both internal consistency (two different starting structures and four different trajectories lead to a consistent average structure) and considerable agreement with the X-ray crystal structure of this sequence and NMR data on duplex DNA in aqueous solution. This internal consistency of structure for a given sequence suggests that one can now begin to realistically examine sequence-dependent structural effects in DNA duplexes using molecular dynamics.

Base Sequence↗

PubMed1998

Solution structure of a highly stable DNA duplex conjugated to a minor groove binder.

The tripeptide 1,2-dihydro-(3 H )-pyrrolo[3,2- e ]indole-7-carboxylate (CDPI3) binds to the minor groove of DNA with high affinity. When this minor groove binder is conjugated to the 5'-end of short oligonucleotides the conjugates form unusually stable hybrids with complementary DNA and thus may have useful diagnostic and/or therapeutic applications. In order to gain an understanding of the structural interactions between the CDPI3minor groove binding moiety and the DNA, we have determined and compared the solution structure of a duplex consisting of oligodeoxyribonucleotide 5'-TGATTATCTG-3' conjugated at the 5'-end to CDPI3 and its complementary strand to an unmodified control duplex of the same sequence using nuclear magnetic resonance techniques. Thermal denaturation studies indicated that the hybrid of this conjugate with its complementary strand had a melting temperature that was 30 degrees C higher compared with the unmodified control duplex. Following restrained molecular dynamics and relaxation matrix refinement, the solution structure of the CDPI3-conjugated DNA duplex demonstrated that the overall shape of the duplex was that of a straight B-type helix and that the CDPI3moiety was bound snugly in the minor groove, where it was stabilized by extensive van der Waal's interactions.

DNA↗

PubMedDate not supplied

(Aminomethyl)phosphonate derivatives of oligonucleotides.

Oligothymidylate (aminomethyl)phosphonates have been prepared, and their enzymatic and physicochemical properties have been studied. The individual isomers of the protected dimers have been separated, characterized, and incorporated into oligonucleotides in which the backbone consists of alternating (aminomethyl)phosphonate and phosphodiester linkages. One of these net neutral, single isomer oligonucleotides forms a duplex with its complementary sequence which is more stable than the corresponding natural counterpart, whereas the other isomer is considerably less stable. Specificity of hybridization is maintained, as determined by the reduction in melting temperature observed upon the introduction of mismatches into the complementary strand of the duplex. The (aminomethyl)-phosphonate linkage is stable toward enzymatic degradation but can be hydrolyzed in aqueous solution at elevated temperature.

Base Sequence↗

PubMedDate not supplied

Introduction of 6-formylcytidine into a Myb binding sequence.

Single 6-formylcytidine was introduced into a oligonucleotide duplex (23 mers) as a substitute for thymidine in the Myb binding sequence of 3'-TTGAC-5'. The modified duplex showed Tm of 67 degrees C, which was six degrees lower than the Tm of the native duplex. Binding affinity of the 23-mers to the Myb protein was estimated by electrophoretic mobility shift assays, and the binding was almost completely abolished.

Binding Sites↗

PubMed1992

Structure and applications of intermolecular DNA triplexes.

Current DNA binding drugs are not sequence specific. Triplex-forming oligonucleotides will bind targeted duplex DNA sites in a sequence-specific manner. A new class of DNA binding molecules based on triple-helical DNA formation promises a sequence-specific method of targeting discrete regions of DNA. DNA modifying molecules linked to third strands have been shown to modify only regions of DNA to which they were targeted. Current research will increase the understanding of triplex DNA structure and will lead to improved DNA binding drugs.

Base Composition↗

PubMed1995

Origins of high sequence selectivity: a stopped-flow kinetics study of DNA/RNA hybridization by duplex- and triplex-forming oligonucleotides.

Stopped-flow UV kinetics and thermal denaturation experiments are used to examine the origins of high sequence selectivity and binding affinity of circular triplex-forming oligonucleotides with single-stranded DNA/RNA targets. These 34-nt probes are hybridized to a series of 12-nt target sequences which are fully complementary or which contain a single mismatch. Also studied for comparison are standard 12-nt Watson-Crick DNA or RNA complements. Several novel findings are described: (1) Circular triplex-forming oligomers bind targets with very high thermodynamic selectivity (up to 8-10 kcal/mol against a single-nucleotide mismatch), while linear strands show only 2-3 kcal/mol selectivity. (2) Rates for triplex formation by circular ligands are much greater than other reported triplex formation modes and are nearly the same as for Watson-Crick duplex formation. (3) DNA-DNA and RNA-RNA hybridization rates are similar for both duplex and triplex formation. (4) For both modes of binding, hybridization rates do not vary when a mismatch is introduced into the target, and, therefore, binding selectivity is reflected in large variations in dissociation, rather than association rates. Finally, (5) binding selectivity of circular ligands becomes significantly greater as pH is lowered; results indicate that the high sequence selectivity of the circular DNA ligand is due in large part to the special stability of the protonated C+G-C triad relative to unprotonated mismatched triads. The results are useful in the understanding of properties of nucleic acid complexes in general and give insight into optimum design for synthetic DNA-binding ligands.

Base Sequence↗

PubMed1992

Cell-free synthesis of the branched RNA-linked msDNA from retron-Ec67 of Escherichia coli.

msDNA-Ec67 is produced in a clinical strain of Escherichia coli and composed of a 67-base single-stranded DNA, which is linked to the 2'-OH group of the 15th rG residue of a 58-base RNA molecule by a 2',5'-phosphodiester linkage (Lampson, B. C., Sun, J., Hsu, M.-Y., Vallejo-Ramirez, J., Inouye, S., and Inouye, M. (1989) Science 243, 1033-1038). The production of msDNA-Ec67 is dependent upon retron-Ec67, which consists of the msr-msd region and the gene for reverse transcriptase (RT). These two elements were separately cloned into plasmids; p67-BHO.6 contained the msr-msd region and pRT-67 contained the RT gene under the lpp-lac promoter-operator. msDNA-Ec67 was produced only when cells were transformed with both plasmids. In addition, msDNA-Ec67 was synthesized in a cell-free system using total RNA prepared from cells harboring plasmid p67-BHO.6 and purified Ec67-RT. Using this cell-free system, the priming reaction, during initiation of DNA synthesis, was demonstrated to be a specific template-directed event; only dTTP was incorporated into a 132-base precursor RNA yielding a 133-base compound. This specific dT addition could be altered to dA or dC by simply substituting the 118th A residue of the putative msr-msd transcript with a T or G residue. The priming reaction was blocked when A was substituted for G at the 15th residue of the precursor RNA transcript, which corresponds to the branched rG residue in msDNA. DNA chain elongation could be terminated by adding ddNTP in the cell-free system, forming a sequence ladder. The DNA sequence determined from this ladder completely agreed with the msDNA sequence. The RT extension reaction was completely blocked when the RNA preparation was treated with RNase A but not when the preparation was treated with DNase. This clearly demonstrates that RNA but not DNA is responsible for the msDNA production. A part of the fully extended cell-free product contained a 13-base RNA strand resistant to RNase A, which is consistent with the previously proposed model. In this model, the 5'-end sequence of the msr-msd transcript (a2; bases 1-13) forms a duplex with the 3'-end sequence (a1) of the same transcript, thus serving as a primer, as well as a template for msDNA synthesis by RT. Our results are inconsistent with a model recently proposed by Lease and Yee (Lease, R. A., and Yee, T. (1991) J. Biol. Chem. 266, 14497-14503).

Base Sequence↗

PubMed2003

Characterization of ciprofloxacin binding to the linear single- and double-stranded DNA.

The binding of ciprofloxacin to natural and synthetic polymeric DNAs was investigated at different solvent conditions using a combination of spectroscopic and hydrodynamic techniques. In 10 mM cacodylate buffer (pH 7.0) containing 108.6 mM Na(+), no sequence preferences in the interaction of ciprofloxacin with DNA was detected, while in 2 mM cacodylate buffer (pH 7.0) containing only 1.7 mM Na(+), a significant binding of ciprofloxacin to natural and synthetic linear double-stranded DNA was observed. At low ionic strength of solution, ciprofloxacin binding to DNA duplex containing alternating AT base pairs is accompanied by the largest enhancement in thermal stability (e.g. DeltaT(m) approximately 10 degrees C for poly[d(AT)].poly[d(AT)]), and the most pronounced red shift in the position of the maximum of the fluorescence emission spectrum (lambda(max)). Similar red shift in the position of lambda(max) is also observed for ciprofloxacin binding to dodecameric duplex containing five successive alternating AT base pairs in the row. On the other hand, ciprofloxacin binding to poly[d(GC)].poly[d(GC)], calf thymus DNA and dodecameric duplex containing a mixed sequence is accompanied by the largest fluorescence intensity quenching. Addition of NaCl does not completely displace ciprofloxacin bound to DNA, indicating the binding is not entirely electrostatic in origin. The intrinsic viscosity data suggest some degree of ciprofloxacin intercalation into duplex.

Animals↗

PubMed2001

PD-loop technology: PNA openers at work.

A concise survey of the emerging PD-loop technology is presented, which outlines several exemplary methods with robust DNA diagnostic potential: duplex DNA capture, topological DNA labeling, nondenaturing DNA sequencing and hybridization of molecular beacons to double-stranded DNA. Advantages of these new PNA-based assays over existing techniques for sequence-specific detection and manipulation of DNA duplexes are discussed. Future prospects for the further development of PD-loop technology are highlighted.

Base Sequence↗

PubMed2001

Protein-free parallel triple-stranded DNA complex formation.

A 14 nt DNA sequence 5'-AGAATGTGGCAAAG-3' from the zinc finger repeat of the human KRAB zinc finger protein gene ZNF91 bearing the intercalator 2-methoxy,6-chloro,9-amino acridine (Acr) attached to the sugar-phosphate backbone in various positions has been shown to form a specific triple helix (triplex) with a 16 bp hairpin (intramolecular) or a two-stranded (intermolecular) duplex having the identical sequence in the same (parallel) orientation. Intramolecular targets with the identical sequence in the antiparallel orientation and a non-specific target sequence were tested as controls. Apparent binding constants for formation of the triplex were determined by quantitating electrophoretic band shifts. Binding of the single-stranded oligonucleotide probe sequence to the target led to an increase in the fluorescence anisotropy of acridine. The parallel orientation of the two identical sequence segments was confirmed by measurement of fluorescence resonance energy transfer between the acridine on the 5'-end of the probe strand as donor and BODIPY-Texas Red on the 3'-amino group of either strand of the target duplex as acceptor. There was full protection from OsO(4)-bipyridine modification of thymines in the probe strand of the triplex, in accordance with the presumed triplex formation, which excluded displacement of the homologous duplex strand by the probe-intercalator conjugate. The implications of these results for the existence of protein-independent parallel triplexes are discussed.

2,2'-Dipyridyl↗

PubMed1995

Solution conformation of [AF]dG opposite a -1 deletion site in a DNA duplex: intercalation of the covalently attached aminofluorene ring into the helix with base displacement of the C8-modified Syn guanine into the major groove.

This paper reports on the solution structure of the [AF]dG adduct positioned opposite a deletion site in a DNA oligomer duplex that defines the alignment of the covalent aminofluorene--C8-guanine adduct relative to the deletion site. The combined NMR molecular mechanics computational studies were undertaken on the [AF]dG adduct embedded in the d(C5-[AF]G6-C7).d(G16-G17) sequence context in a duplex containing 11 residues on the modified strand and 10 on the partner, with no base opposite the modification. The exchangeable and nonexchangeable protons of the aminofluorene moiety and the nucleic acid were assigned following analysis of two-dimensional NMR data sets in H2O and D2O solution. The solution conformation of the [AF]G.del 11-mer duplex has been determined by incorporating intramolecular and intermolecular proton-proton distances defined by lower and upper bounds deduced from NOESY spectra as restraints in molecular mechanics computations in torsion angle space. The aminofluorene ring of [AF]dG6 is intercalated between intact Watson-Crick dC5.dG17 and dC7.dG16 base pairs with the guanine base of [AF]dG6 in a syn alignment displaced into the major groove. The syn glycosidic torsion angle at [AF]dG6 is supported by both carbon and proton chemical shift data for the sugar resonances of the modified guanine residue. The long axis of the aminofluorene ring is parallel to the long axis of the flanking dG.dC base pairs with the AF ring undergoing rapid 180 degrees flips on the NMR time scale. The intercalation site is wedge shaped with a pronounced propeller-twisting and buckling of the dC5.dG17 base pair. The guanine base of [AF]dG6, which is positioned in the major groove, is inclined relative to the helix axis and stacks over the 5'-flanking dC5 residue in the solution structure. The intercalative-base displacement structure of the [AF]dG.del 11-mer duplex exhibits several unusually shifted proton resonances that can be readily accounted for by the ring current contributions of the guanine purine and carcinogen fluorene aromatic rings of the [AF]dG6 adduct. There are similarities between this structure of the AF-C8-dG covalent adduct positioned opposite a deletion site and the (+)-trans-anti-BP-N2-dG covalent adduct positioned opposite a deletion site in the same sequence context reported previously from this laboratory [Cosman et al. (1994) Biochemistry 33, 11507-11517]. The chromophores are intercalated into the helix opposite the deletion site with displacement of the modified guanine into the major groove in both cases.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗

PubMed2000

Monoalkylation of DNA by reductively activated FR66979.

The antitumor antibiotic FR66979 has previously been shown to form interstrand cross-links in duplex DNA at the sequence [5'-d(CG)]2, linking the exocyclic amino groups (N2) of deoxyguanosine (dG) residues. During the reaction of reductively activated FR66979 with DNA. products are formed which have electrophoretic mobility in denaturing polyacrylamide gels which is intermediate between that of unmodified and interstrand cross-linked DNA. We show here that these products are monoadducts between FR66979 and DNA and provide strong evidence for the site of alkylation being N2 of dG. Moreover, the sequence selectivity of monoalkylation reactions between FR66979 and DNA containing either 5'-d(CG).5'-d(CI) or [5'-d(CG)]2 was observed to be ca. 5-fold less than for the related antitumor antibiotic mitomycin C (MC). The mechanistic implications of this result are discussed. Furthermore, it was demonstrated that contrary to a previous report, FR66979 requires DNA to be in duplex form for efficient monoadduct formation.

Alkylation↗

PubMed2006

Sequence-dependent formation of intrastrand crosslink products from the UVB irradiation of duplex DNA containing a 5-bromo-2'-deoxyuridine or 5-bromo-2'-deoxycytidine.

The replacement of thymidine with 5-bromo-2'-deoxyuridine (BrdU) is well-known to sensitize cells to ionizing radiation and photoirradiation. We reported here the sequence-dependent formation of intrastrand crosslink products from the UVB irradiation of duplex oligodeoxynucleotides harboring a BrdU or its closely related 5-bromo-2'-deoxycytidine (BrdC). Our results showed that two types of crosslink products could be induced from d(BrCG), d(BrUG), d(GBrU), or d(ABrU); the C(5) of cytosine or uracil could be covalently bonded to the N(2) or C(8) of its neighboring guanine, and the C(5) of uracil could couple with the C(2) or C(8) of its neighboring adenine. By using those crosslink product-bearing dinucleoside monophosphates as standards, we demonstrated, by using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), that all the crosslink products described above except d(G[N(2)-5]U) and d(G[N(2)-5]C) could form in duplex DNA. In addition, LC-MS/MS quantification results revealed that both the nature of the halogenated pyrimidine base and its 5' flanking nucleoside affected markedly the generation of intrastrand crosslink products. The yields of crosslink products were much higher while the 5' neighboring nucleoside was a dG than while it was a dA, and BrdC induced the formation of crosslink products much more efficiently than BrdU. The formation of intrastrand crosslink products from these halopyrimidines in duplex DNA may account for the photosensitizing effects of these nucleosides.

Base Sequence↗