Sequence-specific base pair mimics are efficient topoisomerase IB inhibitors.

Topoisomerase IB controls DNA topology by cleaving DNA transiently. This property is used by inhibitors, such as camptothecin, that stabilize, by inhibiting the religation step, the cleavage complex, in which the enzyme is covalently attached to the 3'-phosphate of the cleaved DNA strand. These drugs are used in clinics as antitumor agents. Because three-dimensional structural studies have shown that camptothecin derivatives act as base pair mimics and intercalate between two base pairs in the ternary DNA-topoisomerase-inhibitor complex, we hypothesized that base pairs mimics could act like campthotecin and inhibit the religation reaction after the formation of the topoisomerase I-DNA cleavage complex. We show here that three base pair mimics, nucleobases analogues of the aminophenyl-thiazole family, once targeted specifically to a DNA sequence were potent topoisomerase IB inhibitors. The targeting was achieved through covalent linkage to a sequence-specific DNA ligand, a triplex-forming oligonucleotide, and was necessary to position and keep the nucleobase analogue in the cleavage complex. In the absence of triplex formation, only a weak binding to the DNA and topoisomerase I-mediated DNA cleavage was observed. The three compounds were equally active once conjugated, implying that the intercalation of the nucleobase upon triplex formation is the essential feature for the inhibition activity.

Structural basis of the RNase H1 activity on stereo regular borano phosphonate DNA/RNA hybrids.

Numerous DNA chemistries for improving oligodeoxynucleotide (ODN)-based RNA targeting have been explored. The majority of the modifications render the ODN/RNA target insensitive to RNase H1. Borano phosphonate ODN's are among the few modifications that are tolerated by RNase H1. To understand the effect of the stereochemistry of the BH(3) modification on the nucleic acid structure and RNase H1 enzyme activity, we have investigated two DNA/RNA hybrids containing either a R(P) or S(P) BH(3) modification by nuclear magnetic resonance (NMR) spectroscopy. T(M) studies show that the stabilities of R(P) and S(P) modified DNA/RNA hybrids are essentially identical (313.8 K) and similar to that of an unmodified control (312.9 K). The similarity is also reflected in the imino proton spectra. To characterize such similar structures, we used a large number of NMR restraints (including dipolar couplings and backbone torsion angles) to determine structural features that were important for RNase H1 activity. The final NMR structures exhibit excellent agreement with the data (total R(x) values of <6%) with helical properties between those of an A and B helix. Subtle backbone variations are observed in the DNA near the modification, while the RNA strands are relatively unperturbed. In the case of the S(P) modification, for which more perturbations are recorded, a slightly narrower minor groove is also obtained. Unique NOE base contacts localize the S(P) BH(3) group in the major groove while the R(P) BH(3) group points away from the DNA. However, this creates a potential clash of the R(P) BH(3) groups with important RNase H1 residues in a complex, while the S(P) BH(3) groups could be tolerated. We therefore predict that on the basis of our NMR structures a fully R(P) BH(3) DNA/RNA hybrid would not be a substrate for RNase H1.

A rare nucleotide base tautomer in the structure of an asymmetric DNA junction.

The single-crystal structure of a DNA Holliday junction assembled from four unique sequences shows a structure that conforms to the general features of models derived from similar constructs in solution. The structure is a compact stacked-X form junction with two sets of stacked B-DNA-type arms that coaxially stack to form semicontinuous duplexes interrupted only by the crossing of the junction. These semicontinuous helices are related by a right-handed rotation angle of 56.5 degrees, which is nearly identical to the 60 degree angle in the solution model but differs from the more shallow value of approximately 40 degrees for previous crystal structures of symmetric junctions that self-assemble from single identical inverted-repeat sequences. This supports the model in which the unique set of intramolecular interactions at the trinucleotide core of the crossing strands, which are not present in the current asymmetric junction, affects both the stability and geometry of the symmetric junctions. An unexpected result, however, is that a highly wobbled A.T base pair, which is ascribed here to a rare enol tautomer form of the thymine, was observed at the end of a CCCC/GGGG sequence within the stacked B-DNA arms of this 1.9 A resolution structure. We suggest that the junction itself is not responsible for this unusual conformation but served as a vehicle for the study of this CG-rich sequence as a B-DNA duplex, mimicking the form that would be present in a replication complex. The existence of this unusual base lends credence to and defines a sequence context for the "rare tautomer hypothesis" as a mechanism for inducing transition mutations during DNA replication.

Base pairing configuration and stability of an oligonucleotide duplex containing a 5-chlorouracil-adenine base pair.

Inflammation-mediated reactive molecules can damage DNA by oxidation and chlorination. The biological consequences of this damage are as yet incompletely understood. In this paper, we have constructed oligonucleotides containing 5-chlorouracil (ClU), one of the known inflammation damage products. The thermodynamic stability, base pairing configuration, and duplex conformation of oligonucleotides containing ClU paired opposite adenine have been examined. NMR spectra reveal that the ClU-A base pair adopts a geometry similar to that of the T-A base pair, and the ClU-A base pair-containing duplex adopts a normal B-form conformation. The line width of the imino proton of the ClU residue is substantially greater than that of the corresponding T imino proton; however, this difference is not attributed to a reduced thermal or thermodynamic stability or to an increased level of proton exchange with solvent. While the NMR studies reveal an increased level of chemical exchange for the ClU imino proton of the ClU-A base pair, the ClU residue is not a target for removal by the Escherichia coli mispaired uracil glycosylase, which senses damage-related helix instability. The results of this study are consistent with previous reports indicating that the DNA of replicating cells can tolerate substantial substitution with ClU. The fraudulent, pseudo-Watson-Crick ClU-A base pair is sufficiently stable to avoid glycosylase removal and, therefore, might constitute a persistent form of cellular DNA damage.

Conformational heterogeneity and quasi-static self-quenching in DNA containing a fluorescent guanine analogue, 3MI or 6MI.

Two different microenvironments in the DNA sequence 5'-act aGa gat ccc tca gac cct ttt agt cag tGt gga-3' (in both single- and double-stranded forms) are explored using two similar fluorescent nucleoside analogues, 3MI and 6MI. Each probe was evaluated in two environments, one strand with the probe flanked by thymines (PTRT) and the other by adenines (PTRA) with positions indicated by G's in the sequence. Both time-resolved anisotropies and lifetimes of the probes depend upon local interactions, and these are altered by duplex formation. Integrals of lifetime curves compared with quantum yields reveal that each probe displays a "dark" component (below detection limits, with a lifetime of <70 ps). For 6MI in PTRA, this QSSQ "quasi-static self-quenching" or "dark" component represents approximately half the molecules, whether in single- or double-stranded form. In PTRT, 6MI displays an unusual increase in the quantum yield upon formation of the double strand (from 0.107 to 0.189) apparently the result of escape from QSSQ which simultaneously declines from 66 to 33%. This is also accompanied by doubling of steady-state anisotropy. Only 6MI in the PTRT duplex displays a rotational correlation time of >7 ns. In other words, the DS 6MI PTRA environment fails to constrain local motion and QSSQ remains the same as in the single strand; in contrast, the flanking T duplex environment restricts local motion and halves QSSQ. We collected both steady-state and time-resolved fluorescence quenching titrations of 3MI and 6MI in solution with the mononucleotides AMP, CMP, GMP, and TMP. The dynamic quenching rank of the free probes (quenching constant, kq: T > A > G > C) is totally different from that of incorporated probes. We hypothesize the production of weak 3MI.C or 6MI.C complexes that are somehow rendered less subject to dynamic quenching by collision with subsequent C molecules.

Photoaffinity labeling reveals nuclear proteins that uniquely recognize cisplatin-DNA interstrand cross-links.

The DNA-binding inorganic compound cisplatin is one of the most successful anticancer drugs. The detailed mechanism by which cells recognize and process cisplatin-DNA damage is of great interest. Although the family of proteins that bind cisplatin 1,2- and 1,3-intrastrand cross-links has been identified, much less is known about cellular protein interactions with cisplatin interstrand cross-links (ICLs). In order to address this question, a photoreactive analogue of cisplatin, PtBP(6), was used to construct a DNA duplex containing a site-specific platinum ICL. This DNA probe was characterized and used in photo-cross-linking experiments to separate and identify nuclear proteins that bind to the ICL by peptide mass fingerprint analysis. Several such proteins were discovered, including PARP-1, hMutSbeta, DNA ligase III, XRCC1, and PNK. The photo-cross-linking approach was independently validated by an electrophoretic mobility shift assay demonstrating hMutSbeta binding to a cisplatin ICL. Proteins that recognize the platinum ICL were also identified in cisplatin-resistant cells, cells halted at various phases of the cell cycle, and in different carcinoma cells. Nuclear proteins that bind to the platinum ICL differ from those binding to intrastrand cross-links, indicating different mechanisms for disruption of cellular functions.

Kinetic hybrid i-motifs: intercepting DNA with RNA to form a DNA(2)-RNA(2) i-motif.

We have constructed and characterized a long-lived hybrid DNA(2)-RNA(2) i-motif that is kinetically formed by mixing equivalent amount of C-rich RNA (R) and C-rich DNA (D). Circular dichroism shows that these hybrids are distinct from their parent DNA(4) or RNA(4) i-motif. pH dependent CD and UV thermal melting experiments showed that the complexes were maximally stable at pH 4.5, the pK(a) of cytosine, consistent with the complex being held by CH(+)-C base pairs. Fluorescence studies confirmed their tetrameric nature and established the relative strand polarities of the RNA and DNA strands in the complex. These showed that in a hybrid D(2)R(2) i-motif two DNA strands occupy one narrow groove and the two RNA strands occupy the other. This suggests that even the sugar-sugar interactions are highly specific. Interestingly, this hybrid slowly disproportionates into DNA(4) i-motifs and ssRNA which would be valuable to study intermediates in DNA(4) i-motif formation.

Formation of bimetallic Ag-Au nanowires by metallization of artificial DNA duplexes.

Uniform bimetallic nanowires, tunable in size, have been grown on artificial DNA templates via a two-step metallization process. Alkyne-modified cytosines were incorporated into 900-base-pair polymerase-chain-reaction fragments. The alkyne modifications serve as addressable metal-binding sites after conversion to a sugar triazole derivative via click chemistry. Reaction of the Tollens reagent with these sugar-coated DNA duplexes generates Ag0 metallization centers around the sugar modification sites of the DNA. After a subsequent enhancement step using gold, nanowires < or = 10 nm in diameter with a homogeneous surface profile were obtained. Furthermore, the advantage of this two-step procedure lies in the high selectivity of the process, due to the exact spatial control of modified DNA base incorporation and hence the confinement of metallization centers at addressable sites. Besides experiments on a membrane as a proof for the selectivity of the method, atomic force microscopy (AFM) studies of the wires produced on Si-SiO2 surfaces are discussed. Furthermore, we demonstrate time-dependent metallization experiments, monitored by AFM.

Improved delivery in cell culture of radiolabeled antisense DNAs by duplex formation.

PURPOSE: Delivery remains an unresolved problem in applications requiring intravenous administration of DNAs. Recently improved antisense translation interruption in cells was reported for an antisense (AS) oligomer as a duplex compared to singlet AS oligomer presumably because of improved delivery. The unstable phosphodiester backbone of the sense (S) oligomer and its shorter chain length apparently encouraged intracellular dissociation and release of the AS oligomer. We have investigated the mechanism involved to evaluate whether the approach may be useful for antisense radionuclide imaging. PROCEDURES: Duplexes were formed between an AS phosphorothioate DNA against the mdr1 mRNA and the uniform phoshorothioate or uniform phosphodiester sense (S) DNAs with either four or six mismatches. RESULTS: Accumulations in KB-G2 (Pgp++) cells of radiolabeled AS DNA as duplex accumulated threefold higher compared to singlet. Accumulation was still antisense as shown by reduced accumulations with the radiolabel on the S DNA. However, the DNA backbone had no clear influence on accumulations. CONCLUSIONS: Targeting of mRNAs with radiolabeled AS DNAs may be improved in cell culture if duplexed with an S DNA engineered for low hybridization affinity to encourage dissociation in the presence of the target mRNA.

Efficient construction of long DNA duplexes with internal non-Watson-Crick motifs and modifications.

We have developed a semi-synthetic approach for preparing long stretches of DNA (>100 bp) containing internal chemical modifications and/or non-Watson-Crick structural motifs which relies on splint-free, cell-free DNA ligations and recycling of side-products by non-PCR thermal cycling. A double-stranded DNA PCR fragment containing a polylinker in its middle is digested with two restriction enzymes and a small insert ( approximately 20 bp) containing the modification or non-Watson-Crick motif of interest is introduced into the middle. Incorrect products are recycled to starting materials by digestion with appropriate restriction enzymes, while the correct product is resistant to digestion since it does not contain these restriction sites. This semi-synthetic approach offers several advantages over DNA splint-mediated ligations, including fewer steps, substantially higher yields ( approximately 60% overall yield) and ease of use. This method has numerous potential applications, including the introduction of modifications such as fluorophores and cross-linking agents into DNA, controlling the shape of DNA on a large scale and the study of non-sequence-specific nucleic acid-protein interactions.

Structures of the mismatched duplex d(GGGTGCCC) and one of its Watson-Crick analogues d(GGGCGCCC).

The mismatched duplex d(GGGTGCCC) (I) and its two Watson-Crick analogues (dGGGCGCCC) (II) and d(GGGTACCC) (III) were synthesized. The X-ray crystal structures of (I) and (II) were determined at resolutions of 2.5 and 1.7 A (1 A = 0.1 nm) and refined to R factors of 15 and 16%, respectively. (I) and (II) crystallize as A-DNA doublehelical octamers in space groups P61 and P4(3)2(1)2, respectively, and are stable at room temperature. The central two G.T mispairs of (I) adopt the wobble geometry as observed in other G.T mismatches. The two structures differ significantly in their local conformational features at the central helical regions as well as in some global ones. In particular, T-G adopts a large helical twist (44 degrees) whereas C-G adopts a small one (24 degrees). This difference can be rationalized on the basis of simple geometrical considerations. Base-pair stacking energies which were calculated for the two duplexes indicate that (I) is destabilized with respect to (II). Helix-coil transition measurements were performed for each of the three oligomers by means of ultraviolet absorbance spectrophotometry. The results indicate that the stability of the duplexes and the co-operativity of the transition are in the following order: (I) less than (III) less than (II). Such studies may help in understanding why certain regions of DNA are more likely to undergo spontaneous mutations than others.

Endonuclease VII of phage T4 triggers mismatch correction in vitro.

The reactivity of endonuclease VII (gp49 of phage T4) with DNA-loops of eight, four, or one nucleotide, or any of 12 possible base mismatches was tested in vitro. Endonuclease VII introduces double-strand breaks by nick and counter-nick within six nucleotides 3' from the mispairings. High relative cleavage efficiencies at mismatches in heteroduplexes correlate with their decreased thermal stability and vice versa. A delay between nick and counter-nick was sufficient to allow T4 DNA-polymerase and T4 DNA-ligase to correct a C/C-mismatch in vitro, thereby saving the DNA from double-strand breakage. Very short repair tracks of three to four nucleotides mapped between the mismatch and one of the formerly induced nicks, which were subsequently sealed by DNA ligase.

Rapid detection of exon 1 NRAS gene mutations using universal heteroduplex generator technology.

Specific NRAS oncogene missense mutations have been frequently found in some tumors and several hematological diseases, especially in those of myeloid origin. There is a wide range of PCR-based methods for screening and detection of NRAS exon 1 single-base substitutions. However, there are disadvantages and ambiguities associated with these techniques because all of them require either separate probes, separate PCR amplifications, or complicated post-PCR manipulations. This report describes a new approach for detection of NRAS gene mutations at codon 12 and 13 based on the DNA heteroduplex analysis method. The strategy relies upon differential electrophoretic behavior of induced heteroduplex molecules formed by cross-hybridization of two PCR-amplified species, the sample under analysis and the synthetic universal heteroduplex generator (UHG). The screening of a panel of all codon 12 and 13 NRAS mutant DNA variants indicated that this approach discriminates all 12 relevant mutations. The sensitivity of the method was estimated by a competitive assay where mutant alleles could be detected at a dilution level of 1 to 16 wild-type alleles. This UHG technology was tested on some clinical samples previously studied by PCR-ASO. This methodology is highly specific, sensitive, and achieves an appreciable reduction in workload and time because it requires one PCR amplification followed by polyacrylamide gel electrophoresis in standard conditions. We propose that this new approach may be applied as an alternative strategy for codon 12-13 NRAS mutations and it could be easily incorporated into the range of routine assays performed in oncology laboratories.

DNA duplexes containing photoactive derivatives of 2'-deoxyuridine as photocrosslinking probes for EcoRII DNA methyltransferase-substrate interaction.

EcoRII DNA methyltransferase (M.EcoRII) recognizes the DNA sequence 5'.CC*T/AGG.3' and catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the C5 position of the inner cytosine residue (C*). We obtained several DNA duplexes containing photoactive 5-iodo-2'-deoxyuridine (i(5)dU) or 5-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl]-2'-deoxyuridine (Tfmdp-dU) to characterize regions of M.EcoRII involved in DNA binding and to investigate the DNA double helix conformational changes that take place during methylation. The efficiencies of methylation, DNA binding affinities and M.EcoRII-DNA photocrosslinking yields strongly depend on the type of modification and its location within the EcoRII recognition site. The data obtained agree with the flipping of the target cytosine out of the DNA double helix for catalysis. To probe regions of M.EcoRII involved in DNA binding, covalent conjugates M.EcoRII-DNA were cleaved by cyanogen bromide followed by analysis of the oligonucleotide-peptides obtained. DNA duplexes containing i(5)dU or Tfmdp-dU at the central position of the recognition site, or instead of the target cytosine were crosslinked to the Gly(268)-Met(391) region of the EcoRII methylase. Amino acid residues from this region may take part both in substrate recognition and stabilization of the extrahelical target cytosine residue.

[New non-hydrolyzable substrate analogs for 8-oxoguanine-DNA glycosylases]. / Novye negidrolizuemye analogi substratov 8-oksoguanin-DNK-glikolilaz.

8-Oxoguanine-DNA glycosylases play a key role in the repair of oxidatively damaged DNA. The Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) and human 8-oxoguanine-DNA glycosylase (hOGG1) are DNA base excision repair enzymes that catalyze the removal of 7,8-dihydro-8-oxoguanine (oxoG) residue, and cleave DNA strand. Specific contacts between DNA phosphate groups and amino acids from active centers of these enzymes play a significant role in DNA-protein interactions. In order to design new non-hydrolyzable substrate analogs of Fpg and hOGG1 for structural studies modified DNA duplexes containing pyrophosphate or OEt-substituted pyrophosphate internucleotide (SPI) groups near the damage were tested. We showed that enzymes recognize and specifically bind to DNA duplexes obtained. The mechanism of incision of oxoG by the Fpg and hOGG1 was determined. We revealed that both enzymes were not able to excise the oxoG residue from DNA containing modified phosphates immediately 3' to the oxoG. In contrast, Fpg and hOGG1 effectively incise DNA duplex carrying analogous phosphate modifications 5' to the oxoG. Non-cleavable oxoG-containing DNA duplexes bearing pyrophosphate or substituted pyrophosphate groups immediately 3' to the oxoG are specific inhibitors for both 8-oxoguanine-DNA glycosylases and can be used for structural studies of complexes comprising a oxoG-containing DNA bound to catalytically active wild-type enzymes as well as their pro- and eucaryotic homologs.

[Synthesis and properties of DNA duplexes containing hydrocarbon bridges instead of a nucleoside residue]. / Sintez i svoistva DNK-dupleksov, soderzhashchikh uglevodorodnye mostiki vmesto odnogo iz nukleozidnykh ostatkov.

DNA duplexes 14 bp long containing an EcoRII and MvaI restriction site in which a nucleoside is substituted by 1,3-diaminopropane or 1,3-propanediol residue have been chemically synthesized. Diaminopropane bridge was introduced by the chemical ligation, whereas the oligonucleotide containing propanediol was prepared by automatic solid phase phosphoroamidite method on "Victoria-4M" synthesizer. As CD and UV spectra show, the modification destabilises the duplex by 18-20 degrees C without essential distortion of the double helix, except for increase of the conformational mobility in the modified site.

[A new type of DNA modification. Specific introduction of 3'-5'-pyrophosphate internucleotide bonds]. / Novyi tip modifikatsii DNK. Napravlennoe vvedenie 3'-5'-pirofosfatnykh mezhnukleotidnykh sviazei.

Novel compounds--modified DNA-duplexes containing 3'-5'-pyrophosphate internucleotide bonds--have been synthesized. The synthesis was carried out by means of chemical template-guided polycondensation of 3'-5'-diphosphate of decanucleotide pTGGCCAAGCTp, which forms DNA-like duplexes as a result of self-association. 1-Ethyl-3-(3'-dimethylaminopropyl)carbodiimide was used as a condensing agent. Almost quantitative yield of polymer products was achieved by incubation of the reaction mixture for 72 hours. The pyrophosphate type of the internucleotide bonds was confirmed by complete cleavage of these polymers by trifluoroacetic anhydride. The interaction of DNA-duplexes, containing the 3'-5'-pyrophosphate bonds, with restriction endonucleases BspI, HindIII and AluI was studied. BspI and HindIII were able to hydrolyze modified duplexes giving corresponding decanucleotides, whereas AluI failed to split the duplexes.

[Stabilization of G x C-containing DNA duplexes by polyamides with parallel orientation in the minor groove]. / Stabilizatsiia G x C-soderzhashchikh DNK-dupleksov poliamidami s parallel'noi orientatsiei v maloi borozdke.

The polyamides based on 4-amino-1-methylpyrrol-2-carboxylic acid, 4-amino-1-methylimidazole-2-carboxylic acid, and beta-alanine that stabilize oligonucleotide duplexes consisting of G x C pairs through parallel packing in the minor groove were studied. The initial duplex TTGCGCp x GCGCAA melts at 28 degrees C; the TTGCGCp[NH(CH2)3COPyIm betaImNH(CH2)3NH(CH3)2][NH(CH2)3COIm betaImPyNH(CH2)3N(CH3)2] x GCGCAA duplex (bisphosphoramidate with parallel orientation of ligands, where Py, Im, and beta are the residues of 1-methyl-4-aminopyrrol-2-carboxylic and 1-methyl-4-aminoimidazole-2-carboxylic acids and beta-alanine, respectively), at 48 degrees C; and the TTGCGCp[NH(CH2)3COIm betaImPyNH(CH2)3COIm betaImPyNH(CH2)3N(CH3)2] x GCGCAA duplex (a hairpin structure with antiparallel orientation), at 56 degrees C. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 5; see also http: // www.maik.ru.

The use of hairpin DNA duplexes as HIV-1 fusion inhibitors: synthesis, characterization, and activity evaluation.

Discovery of new drugs for the treatment of AIDS that possess unique structures associated with novel mechanisms of action are of great importance due the rapidity with which drug-resistant HIV-1 strains evolve. Recently we reported on a novel class of DNA duplex-based HIV-1 fusion inhibitors modified with hydrophobic groups. The present study describes a new category of hairpin fusion inhibitor DNA duplexes bearing a 3 nucleotide loop located at either the hydrophobic or hydrophilic end. The new loop structures were designed to link 2 separate duplex-forming oligodeoxynucleotides (ODNs) to make helix-assembly easier and more thermally stable resulting in a more compact form of DNA duplex based HIV-1 fusion inhibitors. A series of new hairpin duplexes were tested for anti-HIV-1 cell-cell membrane fusion activity. In addition, Tm, CD, fluorescent resonance energy transfer assays, and molecular modeling analyses were carried out to define their structural activity relationships and possible mechanisms of action.