Use of oligonucleotides to define the site of interstrand cross-links induced by Adriamycin.

It has been known for several years that Adriamycin forms adducts and interstrand cross-links when reacted for long periods of time with bacterial and mammalian DNA in vitro, with the cross-link being restricted to 2 bp elements containing GpC sequences. The self-complementary 20mer deoxyoligonucleotide TA4T4GCA4T4A has been used in this study as a model of the apparent G-G cross-linking site at GpC sequences. The rate of formation of cross-links, as well as the dependence on both Adriamycin and Fe(III) concentration, were similar with this oligonucleotide as compared with calf thymus DNA. The cross-linking was demonstrated on both denaturing and non-denaturing sequencing gels. The half-life of the G-G cross-link was 40 h, consistent with that implied with high molecular weight, heterogeneous sequence DNA. Exonuclease III digests of adducts formed with 20mer deoxyoligonucleotides containing single, central G-G, G-I and I-I potential cross-links revealed that a guanine residue is required at both ends of the cross-link. No cross-linking was observed with a similar oligonucleotide containing only a single central (G.C) bp.     

DNA modifications by a novel bifunctional trinuclear platinum phase I anticancer agent.

The DNA-binding profile of a novel, trinuclear platinum Phase I clinical agent (BBR3464) is summarized. The structure of BBR3464 is best described as two trans-[PtCl(NH3)2] units linked by a tetra-amine [trans-Pt(NH3)2{H2N(CH2)6NH2}2]2+ unit. The +4 charge of BBR3464, the presence of at least two Pt coordination units capable of binding to DNA, and the consequences of such DNA binding are remarkable departures from the cisplatin structural paradigm. The chemical and biological features argue that the drug should be considered the first clinical representative of an entirely new structural class of DNA-modifying anticancer agents. The high charge on BBR3464 facilitates rapid binding to DNA with a t1/2 of approximately 40 min, significantly faster than the neutral cisplatin. The melting temperature of DNA adducted by BBR3464 increased at low ionic strength but decreased in high salt for the same rb. This unusual behavior is in contrast to that of cisplatin. BBR3464 produces an unwinding angle of 14 degrees in negatively supercoiled pSP73 plasmid DNA, indicative of bifunctional DNA binding. Quantitation of interstrand DNA-DNA cross-linking in plasmid pSP73 DNA linearized by EcoRI indicated approximately 20% of the DNA to be interstrand cross-linked. While this is significantly higher than the value for cisplatin, it is, interestingly, lower than that for dinuclear platinum compounds such as [{trans-PtCl(NH3)2}2H2N(CH2)6NH2]2+ (BBR3005) where interstrand cross-linking efficiency may be as high as 70-90%. Either the presence of charge in the linker backbone or the increased distance between platinating moieties may contribute to this relatively decreased ability of BBR3464 to induce DNA interstrand cross-linking. Fluorescence experiments with ethidium bromide were consistent with the formation of long-range delocalized lesions on DNA produced by BBR3464. The sequence preference for BBR3464 on plasmid DNA was determined to the exact base pair by assaying extension of the polynucleotide by VentR(exo+) DNA polymerase. Strong sequence preference for single dG or d(GG) sites was suggested. The presence of relatively few blocks on DNA in comparison to either cisplatin or BBR3005 was indicative of high sequence selectivity. The following appropriate sequence where stop sites occur was chosen: [sequence: see text] molecular modeling on 1,4 interstrand (G'30 to G33) and 1,5 intrastrand (G33 to G29) cross-links further confirmed the similarity in energy between the two forms of cross-link. Finally, immunochemical analysis confirmed the unique nature of the DNA adducts formed by BBR3464. This analysis showed that antibodies raised to cisplatin-adducted DNA did not recognize DNA modified by BBR3464. In contrast, DNA modified by BBR3464 inhibited the binding of antibodies raised to transplatin-adducted DNA. Thus, the bifunctional binding of BBR3464 contains few similarities to that of cisplatin but may have a subset of adducts recognized as being similar to the transplatinum species. In summary, the results point to a unique profile of DNA binding for BBR3464, strengthening the original hypothesis that modification of DNA binding in manners distinct from that of cisplatin will also lead to a distinct and unique profile of antitumor activity.     

4-vinyl-substituted pyrimidine nucleosides exhibit the efficient and selective formation of interstrand cross-links with RNA and duplex DNA

The formation of interstrand cross-links in nucleic acids can have a strong impact on biological function of nucleic acids; therefore, many cross-linking agents have been developed for biological applications. Despite numerous studies, there remains a need for cross-linking agents that exhibit both efficiency and selectivity. In this study, a 4-vinyl-substituted analog of thymidine (T-vinyl derivative) was designed as a new cross-linking agent, in which the vinyl group is oriented towards the Watson–Crick face to react with the amino group of an adenine base. The interstrand cross-link formed rapidly and selectively with a uridine on the RNA substrate at the site opposite to the T-vinyl derivative. A detailed analysis of cross-link formation while varying the flanking bases of the RNA substrates indicated that interstrand cross-link formation is preferential for the adenine base on the 5′-side of the opposing uridine. In the absence of a 5′-adenine, a uridine at the opposite position underwent cross-linking. The oligodeoxynucleotides probe incorporating the T-vinyl derivative efficiently formed interstrand cross-links in parallel-type triplex DNA with high selectivity for dA in the homopurine strand. The efficiency and selectivity of the T-vinyl derivative illustrate its potential use as a unique tool in biological and materials research.     

Melting of Cross-Linked DNA V. Cross-Linking Effect Caused by Local Stabilization of the Double Helix

Abstract

DNA interstrand cross-links are usually formed due to bidentate covalent or coordination binding of a cross-linking agent to nucleotides of different strands. However interstrand linkages can be also caused by any type of chemical modification that gives rise to a strong local stabilization of the double helix. These stabilized sites conserve their helical structure and prevent local and total strand separation at temperatures above the melting of ordinary AT and GC base pairs. This local stabilization makes DNA melting fully reversible and independent of strand concentration like ordinary covalent interstrand cross-links. The stabilization can be caused by all the types of chemical modifications (interstrand cross-links, intrastrand cross-links or monofunctional adducts) if they give rise to a strong enough local stabilization of the double helix. Our calculation demonstrates that an increase in stability by 25 to 30 kcal in the free energy of a single base pair of the double helix is sufficient for this “cross-linking effect” (i.e. conserving the helicity of this base pair and preventing strand separation after melting of ordinary base pairs). For the situation where there is more then one stabilized site in a DNA duplex (e.g., 1 stabilized site per 1000 bp), a lower stabilization per site is sufficient for the “cross-linking effect” (18–20 kcal). A substantial increase in DNA stability was found in various experimental studies for some metal-based anti-tumor compounds. These compounds may give rise to the effect described above. If ligand induced stabilization is distributed among several neighboring base pairs, a much lower minimum increase per stabilized base pair is sufficient to produce the cross-linking effect (1 bp- 24.4 kcal; 5 bp- 5.3 kcal; 10 bp- 2.9 kcal, 25 bp- 1.4 kcal; 50 bp- 1.0 kcal). The relatively weak non-covalent binding of histones or protamines that cover long regions of DNA (20–40 bp) can also cause this effect if the salt concentration of the solution is sufficiently low to cause strong local stabilization of the double helix. Stretches of GC pairs more than 25 bp in length inserted into poly(AT) DNA also exhibit properties of stabilizing interstrand cross-links.     

Melting of cross-linked DNA v. cross-linking effect caused by local stabilization of the double helix

DNA interstrand cross-links are usually formed due to bidentate covalent or coordination binding of a cross-linking agent to nucleotides of different strands. However interstrand linkages can be also caused by any type of chemical modification that gives rise to a strong local stabilization of the double helix. These stabilized sites conserve their helical structure and prevent local and total strand separation at temperatures above the melting of ordinary AT and GC base pairs. This local stabilization makes DNA melting fully reversible and independent of strand concentration like ordinary covalent interstrand cross-links. The stabilization can be caused by all the types of chemical modifications (interstrand cross-links, intrastrand cross-links or monofunctional adducts) if they give rise to a strong enough local stabilization of the double helix. Our calculation demonstrates that an increase in stability by 25 to 30 kcal in the free energy of a single base pair of the double helix is sufficient for this "cross-linking effect" (i.e. conserving the helicity of this base pair and preventing strand separation after melting of ordinary base pairs). For the situation where there is more then one stabilized site in a DNA duplex (e.g., 1 stabilized site per 1000 bp), a lower stabilization per site is sufficient for the "cross-linking effect" (18 - 20 kcal). A substantial increase in DNA stability was found in various experimental studies for some metal-based anti-tumor compounds. These compounds may give rise to the effect described above. If ligand induced stabilization is distributed among several neighboring base pairs, a much lower minimum increase per stabilized base pair is sufficient to produce the cross-linking effect (1 bp- 24.4 kcal; 5 bp- 5.3 kcal; 10 bp- 2.9 kcal, 25 bp- 1.4 kcal; 50 bp- 1.0 kcal). The relatively weak non-covalent binding of histones or protamines that cover long regions of DNA (20- 40 bp) can also cause this effect if the salt concentration of the solution is sufficiently low to cause strong local stabilization of the double helix. Stretches of GC pairs more than 25 bp in length inserted into poly(AT) DNA also exhibit properties of stabilizing interstrand cross-links.     

Facile SNP detection using bifunctional, cross-linking oligonucleotide probes

A facile, sensitive method for detecting specific sequences of oligonucleotides was developed. Detection of DNA sequences with single nucleotide discrimination is achieved by combining the selectivity of hybridization with an efficient cross-linking reaction. Readily synthesized bifunctional oligonucleotide probes containing a modified pyrimidine that is capable of forming interstrand cross-links under mild oxidative conditions internally, and biotin at their 5′-termini were used to discriminate between 16-nt long sites in plasmid DNA that differ by a single nucleotide. The target sequence was detected via fluorescence spectroscopy by utilizing conjugates of avidin and horseradish peroxidase in a microtiter plate assay. The method is able to detect as little as 250 fmol of target without using PCR and exhibits single nucleotide discrimination that approaches 200:1. In principle, this method is capable of probing any target sequence containing a 2′-deoxyadenosine.     

Sequence-specific DNA interstrand cross-linking by an aziridinomitosene in the absence of exogenous reductant

The aziridinomitosene derivative (1S,2S)-6-desmethyl(methylaziridino)mitosene (4) was shown to alkylate plasmid DNA at pH 7.4 in the absence of a reducing agent [Vedejs, E., Naidu, B. N., Klapars, A., Warner, D. L., Li, V. -s., Na, Y., and Kohn, H. (2003) J. Am. Chem. Soc. 125, 15796-15806], an activity not found in the parent mitomycins. We sought to evaluate aziridinomitosene 4 for the presence of DNA interstrand cross-linking activity using nonreductive reaction conditions. Radiolabeled DNA treated with 4 was analyzed by denaturing polyacrylamide gel electrophoresis (DPAGE), a technique that readily separates the less mobile cross-linked ds DNA from the more mobile ss DNA products. Nonreduced 4 produced an interstrand cross-link (ICL) in duplex DNA containing 5'-d(CG) sites, and the yield of ICL was comparable to that obtained from reduced MC under similar conditions. A ds DNA having the central tetranucleotide 5'-d(ACGT) provided the greatest ICL yield from both nonreduced 4 and reduced MC. Substitution of 5'-d(CG) with the inverted sequence 5'-d(GC) completely abolished interstrand cross-linking by 4, revealing 5'-d(CG) as its specific site of ICL formation. Replacement of dG at 5'-d(CG) with 2'-deoxyinosine (dI), which lacks the exocyclic C2 amino group present in dG, also prevented DNA ICL formation by 4, revealing an essential role for the dG C2 amino group in the interstrand cross-linking reaction between 4 and duplex DNA. This report directly demonstrates the presence of bifunctional alkylating activity in a nonreduced aziridinomitosene and clearly shows that unreduced 4 alkylates residues in the minor groove of ds DNA, cross-linking with the same 5'-d(CG) sequence specificity displayed by reduced MC.     

Design and synthesis of the novel cross-linking reagents triggered by the triple helix formation.

In our attempt to new nucleobase analogs capable of interstrand cross-linking, we developed 2-amino-6-vinyl purine analog (1). The oligonucleotides incorporating 1 showed efficient interstrand cross-linking with selectivity toward cytidine at a target site. In this paper, we describe the design of the new cross-linking reagents (2) bearing 2-amino-6-vinyl purine motif, and triplex-directed alkylation with 2 to double-stranded DNA.     

Affinity purification and characterization of human O6-alkylguanine-DNA alkyltransferase complexed with BCNU-treated, synthetic oligonucleotide.

Tumor cells resistant to chloroethylnitrosourea (CENU) therapy contain high levels of O6-alkylguanine DNA-alkyltransferase (GATase), a DNA repair enzyme that aborts DNA interstrand cross-linking by removing CENU-induced O6-alkylguanine adducts. Because the transferase binds covalently to CENU-treated oligonucleotides, we reacted partially purified GATase from cultured human lymphoblasts with a BCNU-treated, 35S-5'-end-labeled, synthetic oligonucleotide designed to have a polyadenylated 3' terminus. Immunoprobing Western blots of this reaction mixture with GATase-specific monoclonal antibody indicated that 25-30% of the transferase became complexed. We purified this complex by affinity chromatography with oligo(dT) cellulose, recovering homogenous material that appeared as a discrete 35-kDa Coomassie blue or silver-stained band after SDS-polyacrylamide gel electrophoresis. Autoradiography and Western immunoblotting confirmed that this band contained both the radiolabeled oligonucleotide and the GATase protein.     

Interstrand cross-linking of DNA by FK317 and its deacetylated metabolites FR70496 and FR157471

FR900482 (1) and FR66979 (2) are structurally novel natural products isolated by Fujisawa in 1987 and have been shown to be highly potent antitumor antibiotics structurally related to the mitomycins. Studies on the mode of action have established that these new agents form covalent DNA interstrand cross-links both in vitro and in vivo as a result of the reactive mitosene intermediate generated upon bioreductive activation. Semisynthetic analogues such as FK973 (3) and FK317 (4) were developed in the search for potentially superior clinical candidates. Although FK317 has been shown to be a potent compound, to date no direct evidence of DNA interstrand cross-link sequence specificity has been reported. In this study, DNA interstrand cross-links were generated by treatment of a synthetic duplex DNA substrate with FK317 (4) and its deacetylated metabolites FR70496 (5) and FR157471 (6). Analysis by gel electrophoresis revealed the formation of orientation isomers displaying electrophoretic mobility vastly greater than the mobilities of those generated from FR900482 (1). Despite these differences, it was established by Fe(II)-EDTA footprinting that FK317 (4) as well as 5 and 6 forms DNA interstrand cross-links within the expected 5'CpG3' step, clearly demonstrating that the phenolic hydrogen in 1 and 2 is not a prerequisite for efficient DNA interstrand cross-linking by the FR class of compounds.     

Sequence preferences of DNA interstrand cross-linking agents: dG-to-dG cross-linking at 5'-CG by structurally simplified analogues of mitomycin C

The nucleotide sequence preferences of the DNA interstrand cross-linking agents dehydroretronecine diacetate (DHRA), 2,3-bis(acetoxymethyl)-1-methylpyrrole (BAMP), dehydromonocrotaline, and dehydroretrorsine were studied by using synthetic DNA duplex fragments and polyacrylamide gel electrophoresis (PAGE). These agents have structural features in common with the reductively activated aziridinomitosene of mitomycin C (MC). Like MC, they preferentially cross-linked DNA duplexes containing the duplex sequence 5'-CG. For DHRA and BAMP interstrand cross-linked DNA duplexes, PAGE analysis of iron(II)-EDTA fragmentation reactions revealed the interstrand cross-links to be deoxyguanosine to deoxyguanosine (dG-to-dG), again analogous to DNA cross-links caused by MC. Unlike MC, DHRA could be shown to dG-to-dG cross-link a 5'-GC sequence. Furthermore, the impact of flanking sequence on the efficiency of interstrand cross-linking at 5'-CG was reduced for BAMP, with 5'-TCGA and 5'-GCGC being equally efficiently cross-linked. Possible origins of the 5'-CG sequence recognition common to all of the agents are discussed. A model is presented in which the transition state for the conversion of monoadducts to cross-links more closely resembles ground-state DNA at 5'-CG sequences.     

An asymmetric C8/C8'-tripyrrole-linked sequence-selective pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimer DNA interstrand cross-linking agent spanning 11 DNA base pairs

A novel sequence-selective extended PBD dimer 4 has been synthesized that binds with high affinity to an interstrand cross-linking site spanning 11 DNA base pairs. Despite its molecular weight (984.07) and length, the molecule has significant DNA interstrand cross-linking potency (approximately 100-fold greater than the clinically used agent melphalan) and sub-micromolar cytotoxicity in a number of tumour cell lines, suggesting that it readily penetrates cellular and nuclear membranes to reach its DNA target.     

Mutation in recR gene of Deinococcus radiodurans and possible involvement of its product in the repair of DNA interstrand cross-links

We previously reported that some Deinococcus radiodurans mutants are sensitive to DNA interstrand cross-linking agents but resistant to UV and gamma-rays. We isolated DNA fragments from a D. radiodurans genomic library which complemented the mitomycin C sensitivity of one of these mutants. One 3.2kb-long fragment contains an open reading frame of approximately 700bp and the deduced amino acid sequence is very homologous to other prokaryotic RecR proteins. This open reading frame in the mitomycin C-sensitive mutant strain contains a frame shift mutation at its carboxyl terminal region. These data suggest that RecR protein plays an important role in the resistance to interstrand cross-links in this bacterium.     

Sequence-selective recognition of duplex DNA through covalent interstrand cross-linking: kinetic and molecular modeling studies with pyrrolobenzodiazepine dimers

Members of a homologous series of pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers with C8-O-(CH(2))(n)-O-C8' diether linkages (n = 3-6 for 2a-d, respectively) have been studied for their ability to interact with oligonucleotide duplexes containing potential target binding sites. The results confirm earlier predictions that the n = 3 analogue (2a, DSB-120) will covalently bind to a 5'-Pu-GATC-Py sequence by cross-linking opposite-strand guanines separated by 2 bp. Preference for this DNA sequence is shown using oligonucleotides with altered bases between and/or flanking these guanines. The more extended PBD dimer 2c (n = 5) can span an extra base pair and cross-link the 5'-Pu-GA(T/A)TC-Py sequence. The ability of each homologue to cross-link linear plasmid DNA has been determined, with a rank order that correlates with the reported order of in vitro cytotoxicity: n = 3 (2a) > n = 5 (2c) > n = 6 (2d) > n = 4 (2b). The n = 3 homologue (2a) is >300-fold more efficient at cross-linking DNA than the clinically used cross-linking agent melphalan under the same conditions. Kinetic studies reveal that the n = 3 and 5 dimers achieve faster cross-linking to plasmid DNA (108 and 81% cross-linking h(-1) microM(-1) at 37 degrees C, respectively), whereas the n = 4 and 6 homologues are significantly less efficient at 10.3 and 23% cross-linking h(-1) microM(-1), respectively. Alternating activity for the odd n and even n dimers is probably due to configurational factors governed by the spatial separation of the PBD subunits and the flexible character of the tethering linkage. Molecular modeling confirms the order of cross-linking reactivity, and highlights the role of linker length in dictating sequence recognition for this class of DNA-reactive agent.     

Interstrand cross-linking reaction in triplexes containing a monofunctional transplatin-adduct.

Our aim was to determine whether a single transplatin monofunctional adduct, either trans-[Pt(NH3)2(dC)Cl]+ or trans-[Pt(NH3)2(dG)Cl]+ within a homopyrimidine oligonucleotide, could further react and form an interstrand cross-link once the platinated oligonucleotide was bound to the complementary duplex. The single monofunctional adduct was located at either the 5' end or in the middle of the platinated oligonucleotide. In all the triplexes, specific interstrand cross-links were formed between the platinated Hoogsteen strand and the complementary purine-rich strand. No interstrand cross-links were detected between the platinated oligonucleotides and non-complementary DNA. The yield and the rate of the cross-linking reaction depend upon the nature and location of the monofunctional adducts. Half-lives of the monofunctional adducts within the triplexes were in the range 2-6 h. The potential use of the platinated oligonucleotides to modulate gene expression is discussed.     

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.     

Alkaline gel electrophoresis of deoxyribonucleic acid photoreacted with trimethylpsoralen: rapid and sensitive detection of interstrand cross-links

Restriction fragments of phage lambda and phi X174 deoxyribonucleic acid (DNA) were photoreacted with 4,5',8-trimethylpsoralen to various extents, and the amount of covalent cross-linking was determined by electron microscopy of the DNA under totally denaturing conditions. The DNA was then analyzed by electrophoresis in alkaline agarose gels. A single cross-link in a DNA molecule produced a large decrease in its electrophoretic mobility. With DNA fragments 0.3--4 kilobase pairs in size, the apparent Mr (molecular weight) of the cross-linked DNA was 2.0 +/- 0.1 times and Mr of the unreacted, single-stranded DNA. A single cross-link in a larger DNA molecule resulted in an even greater increase in apparent Mr. Further cross-linking produced a decrease in the apparent Mr of the DNA, reaching a plateau at a value of 1.4 +/- 0.1 times the Mr of the unreacted, single-stranded DNA over a large range of fragment sizes (0.6--10 kilobase pairs). The apparent Mr of the cross-linked DNA was weakly dependent on the percentage of agarose in the gel. Although highly sensitive to interstrand cross-links the electrophoretic mobilities appeared to be unaffected by low levels of monoadducts (trimethylpsoralen covalently bound to one strand of the DNA). The DNA bandwidths increased by as much as 4-fold at low extents of cross-linking, presumably due to heterogeneity in the locations of the cross-links in the DNA molecules. The bands became sharp again at high levels of reaction. These observations from the basis of a new assay for interstrand DNA cross-links that is both more sensitive and more convenient than previous methods.     

Affinity and sequence specificity of DNA binding and site selection for primer synthesis by Escherichia coli primase

Primase is an essential DNA replication enzyme in Escherichia coli and responsible for primer synthesis during lagging strand DNA replication. Although the interaction of primase with single-stranded DNA plays an important role in primer RNA and Okazaki fragment synthesis, the mechanism of DNA binding and site selection for primer synthesis remains unknown. We have analyzed the energetics of DNA binding and the mechanism of site selection for the initiation of primer RNA synthesis on the lagging strand of the replication fork. Quantitative analysis of DNA binding by primase was carried out using a number of oligonucleotide sequences: oligo(dT)(25) and a 30 bp oligonucleotide derived from bacteriophage G4 origin (G4ori-wt). Primase bound both sequences with moderate affinity (K(d) = 1.2-1.4 x 10(-)(7) M); however, binding was stronger for G4ori-wt. G4ori-wt contained a CTG trinucleotide, which is a preferred site for initiation of primer synthesis. Analysis of DNA binding isotherms derived from primase binding to the oligonucleotide sequences by fluorescence anisotropy indicated that primase bound to DNA as a dimer, and this finding was further substantiated by electrophoretic mobility shift assays (EMSAs) and UV cross-linking of the primase-DNA complex. Dissection of the energetics involved in the primase-DNA interaction revealed a higher affinity of primase for DNA sequences containing the CTG triplet. This sequence preference of primase may likely be responsible for the initiation of primer synthesis in the CTG triplet sites in the E. coli lagging strand as well as in the origin of replication of bacteriophage G4.