Attachment of benzaldehyde-modified oligodeoxynucleotide probes to semicarbazide-coated glass.

Attachment of oligodeoxynucleotides (ODNs) containing benzaldehyde (BAL) groups to semicarbazide-coated glass (SC-glass) slides is described. 5'-BAL-ODNs are prepared using automated DNA synthesis and an acetal-protected BAL phosphoramidite reagent. The hydrophobic protecting group simplifies purification of BAL-ODNs by reverse phase HPLC and is easily removed using standard acid treatment. The electrophilic BAL-ODNs are stable in solution, but react specifically with semicarbazide groups to give semicarbazone bonds. Glass slides were treated with a semicarbazide silane to give SC-glass. BAL-ODNs are coupled to the SC-glass surface by a simple one-step procedure that allows rapid, efficient and stable attachment. Hand-spotted arrays of BAL-ODNs were prepared to evaluate loading density and hybridization properties of immobilized probes. Hybridization to radiolabeled target strands shows that at least 30% of the coupled ODNs were available for hybridization at maximum immobilization density. The array was used to probe single nucleotide polymorphisms in synthetic DNA targets, and PCR products were correctly genotyped using the same macroarray. Application of this chemistry to manufacturing of DNA microarrays for sequence analysis is discussed.

A new strategy of discrimination of a point mutation by tandem of short oligonucleotides.

A new strategy based on the use of cooperative tandems of short oligonucleotide derivatives (TSOD) has been proposed to discriminate a "right" DNA target from a target containing a single nucleotide discrepancy. Modification of a DNA target by oligodeoxyribonucleotide reagents was used to characterize their interaction in the perfect and mismatched complexes. It is possible to detect any nucleotide changes in the binding sites of the target with the short oligonucleotide reagent. In the presence of flanking di-3',5'-N-(2-hydroxyethyl)phenazinium derivatives of short oligonucleotides (effectors) the tetranucleotide alkylating reagent modifies DNA target efficiently and site-specifically only in the perfect complex and practically does not modify it in the mismatched complex. It has been shown that TSOD is much more sensitive tool for the detection of a point mutation in DNA as compared to a longer oligonucleotides.

Targeted mutagenesis of DNA with alkylating RecA assisted oligonucleotides.

Site-specific mutation was demonstrated in a shuttle vector system using nitrogen mustard-conjugated oligodeoxyribonucleotides (ODNs). Plasmid DNA was modified in vitro by ODNs containing all four DNA bases in the presence of Escherichia coli RecA protein. Up to 50% of plasmid molecules were alkylated in the targeted region of the supF gene and mutations resulted upon replication in mammalian cells. ODNs conjugated with either two chlorambucil moieties or a novel tetrafunctional mustard caused interstrand crosslinks in the target DNA and were more mutagenic than ODNs that caused only monoadducts.

Sequence-specific targeting and covalent modification of human genomic DNA.

We compare two techniques which enable selective, nucleotide-specific covalent modification of human genomic DNA, as assayed by quantitative ligation- mediated PCR. In the first, a purine motif triplex-forming oligonucleotide with a terminally appended chlorambucil was shown to label a target guanine residue adjacent to its binding site in 80% efficiency at 0.5 microM. Efficiency was higher in the presence of the triplex-stabilizing intercalator coralyne. In the second method, an oligonucleotide targeting a site containing all four bases and bearing chlorambucil on an interior base was shown to efficiently react with a specific nucleotide in the target sequence. The targeted sequence in these cases was in the DQbeta1*0302 allele of the MHC II locus.

[Nucleic acids interactions with short oligonucleotide derivatives. II. Tandem of short oligonucleotides as highly sensitive system for identification of single base substitutions in target DNA]. / Vzaimodeistvie proizvodnykh korotkikh oligonukleotidov s nykleinovymi kislotami. II. Tandem korotkikh oligonukleotidov--vysokochuvstvitel'naia sistema dlia obnaruzheniia odnobukvennoi zameny v DNK-misheni.

A new approach for modification of target DNAs with tandems of derivatives of short oligonucleotides was suggested that allows highly selective modification of perfect duplexes only. At physiological temperatures, the efficiency of DNA modification by a dodecanucleotide alkylating agent was demonstrated to be the same for both perfect and mismatch-containing duplexes, whereas the tetranucleotide reagent in the presence of two flanking effectors alkylated with high selectivity the target DNA in the perfect duplex only.

RecA-catalyzed, sequence-specific alkylation of DNA by cross-linking oligonucleotides. Effects of length and nonhomologous base substitutions.

Oligodeoxyribonucleotides (ODNs) bearing the reactive nitrogen mustard chlorambucil have been used as sequence-directed affinity labeling reagents to investigate the length and homology requirements for RecA-catalyzed alkylation of double-stranded DNA. The cross-linkage reaction, which takes place at the N-7 position of a targeted complementary strand guanine following strand exchange, was highly sequence specific with both a 272 bp DNA fragment and a linearized plasmid. Alkylation required the ODN to be at least 26 nucleotides long and to possess homology to the target in the vicinity of the modification site. The extent of alkylation was improved by using longer ODNs, with a 50-mer giving over 50% reaction. Mismatches inhibited alkylation when they perturbed the structure of the strand exchange product near the targeted guanine. Longer heterology also inhibited alkylation when it prevented strand exchange. Our inability to detect cross-linkage in stable synaptic complexes unable to undergo complete strand exchange is best explained by a model for homologous alignment in which the presynaptic filament approaches from the minor groove of the duplex. Since the N-7 position of guanine is in the major groove, it is inaccessible to the tethered chlorambucil group of the ODN during the search for homology. The reaction specificity of chlorambucil-bearing ODNs suggests that they may have general use as recombinase-mediated DNA targeting agents.

Rapid and efficient hybridization-triggered crosslinking within a DNA duplex by an oligodeoxyribonucleotide bearing a conjugated cyclopropapyrroloindole.

The antitumor antibiotic CC-1065 binds in the minor groove of double-stranded DNA, and the cyclopropapyrroloindole (CPI) subunit of the drug alkylates adjacent adenines at their N-3 position. We have attached racemic CPI to oligodeoxyribonucleotides (ODNs) via a terminal phosphorothioate at either the 3'- or 5'-end of the ODNs. These conjugates were remarkably stable in aqueous solution at neutral pH even in the presence of strong nucleophiles. When a 3'-CPI-ODN conjugate was hybridized to a complementary DNA strand at 37 degrees C, the CPI moiety alkylated nearby adenine bases of the complement efficiently and rapidly, with a half-life of a few minutes. The 4'-CPR- ODN conjugate showed very little reactivity within the duplex. CPI-ODN conjugates should be highly effective sequence-specific inhibitors of single-stranded viral DNA replication or gene selective inhibitors of transcription initiation.

Sequence-specific covalent modification of DNA by cross-linking oligonucleotides. Catalysis by RecA and implication for the mechanism of synaptic joint formation.

Oligodeoxynucleotides (ODNs) were conjugated to chlorambucil and used as affinity labeling reagents to study joint molecule formation by the Escherichia coli recombinase recA. Chlorambucil is a bifunctional nitrogen mustard which alkylates the N-7 position of guanine in the major groove of double-stranded DNA (dsDNA). Incoming ODNs at least 30 nucleotides long cross-linked to a long homologous duplex DNA in the presence of recA and ATP gamma S. Efficient cross-linkage to the complementary recipient strand of the joint occurred preferentially at guanines positioned 5' relative to the appended chlorambucil group. The pattern of recipient strand alkylation was identical to that observed within a protein-free duplex and indicated that strand exchange had occurred prior to alkylation. Modification of the outgoing homologous strand of the joint was less efficient and spanned a 15-20 nucleotide long region offset to the 3' side of the tethered chlorambucil. Alkylation of both recipient and outgoing strands in the same joint molecule occurred with low frequency. By contrast, no affinity alkylation of the displaced strand was observed within a synthetic D-loop. These reaction patterns suggest that the incoming ODN approaches from the minor groove of the duplex to yield a poststrand exchange joint in which the major groove of the newly formed heteroduplex harbors the outgoing strand in an unpaired state. No evidence was obtained for the involvement of a triple-stranded DNA intermediate in recombination.

[Effect of the intercalating dyes ethidium and phenazine, covalently jointed to the 5'- or 3'-end of the pentanucleotide d(pGAAAG), on the thermodynamics of complementary and cooperative interaction]. / Vliianie interkaliruiushchikh krasitelei étidiia i fenaziniia, kovalentno prisoedinennykh k 5'- ili 3'-kontsu pentanukleotida d(pGAAAG), na termodinamiku komplementarnogo i kooperativnogo vzaimodeistviia.

Thermal stability was studied and thermodynamic parameters of complex formation were calculated for pentanucleotide complexes [formula: see text] and corresponding complexes of pentanucleotide derivatives carrying at their 5'- or 3'-ends covalently bound residues of intercalating dyes: N-(2-hydroxyethyl)-phenazine (Phn) or 2-N-(3-aminopropionyl)-ethidium. Pentanucleotide derivatives were shown to form more stable complementary complexes. The best stabilizing effect was observed when the dye was oriented towards the long single-stranded fragment of tetradecanucleotide, melting temperature of the complexes being by 22.5 (Phn) and 31.2 degrees C (Etd) greater than that of unmodified complexes in the case of 5'-derivatives and by 21.6 (Phn) and 27.2 degrees C (Etd) for 3'-derivatives. Cooperativity constant of pentanucleotide derivatives in "tandem" complexes IV-VII was higher than that of unmodified pentanucleotide. For complex IV at 37 degrees C the constant values were 33 (unmodified), 35 (5'Phn), 57 (3'Phn), 190 (5'Etd), 100 (3'Etd). With n = 3 in complex VII cooperativity constants approached 1 in any case.

Discovery of short, 3'-cholesterol-modified DNA duplexes with unique antitumor cell activity.

A new class of modified oligodeoxynucleotides with unique, selective cytotoxic properties has been discovered. Self-complementary, 3'-cholesterol-modified oligodeoxynucleotides caused morphology changes and death in certain cancer cell lines, whereas other cell lines were unaffected. Susceptible cells were killed in a dose-dependent manner at submicromolar concentrations. Optimum potency was exhibited by phosphodiester duplexes approximately 10 base pairs in length, and base composition was important only in the context of duplex stability. Phosphorothioate analogues were less potent. Although the molecular mechanism of action of these unique compounds is not yet known, they offer potential applications in cancer therapy and in studies of cell death. In addition, the path toward elucidation of the structure-based biological activity of these oligonucleotides should be especially instructive for researchers studying sequence-specific effects.

Synthetic RNA-cleaving molecules mimicking ribonuclease A active center. Design and cleavage of tRNA transcripts.

RNA cleaving molecules were synthesized by conjugating imidazole residues imitating the essential imidazoles in the active center of pancreatic ribonuclease to an intercalating compound, derivative of phenazine capable of binding to the double stranded regions of polynucleotides. Action of the molecules on tRNA was investigated. It was found, that some of the compounds bearing two imidazole residues cleave tRNA under physiological conditions. The cleavage reaction shows a bell-shaped pH dependence with a maximum at pH 7.0 indicating participation of protonated and non-protonated imidazole residues in the process. Under the conditions stabilizing the tRNA structure, a tRNAAsp transcript was cleaved preferentially at the junctions of the stem and loop regions of the cloverleaf tRNA fold, at the five positions C56, C43, C20.1, U13, and U8, with a marked preference for C56. This cleavage pattern is consistent with a hydrolysis mechanism involving non-covalent binding of the compounds to the double-stranded regions of tRNA followed by an attack of the imidazole residues at the juxtaposed flexible single-stranded regions of the molecule. The compounds provide new probes for the investigation of RNA structure in solution and potential reactive groups for antisense oligonucleotide derivatives.

Synthesis and high stability of complementary complexes of N-(2-hydroxyethyl)phenazinium derivatives of oligonucleotides.

Two simple methods for the synthesis of oligonucleotides bearing a N-(2-hydroxyethyl)phenazinium (Phn) residue at the 5'- and/or 3'-terminal phosphate groups are proposed. By forming complexes between a dodecanucleotide d(pApApCpCpTpGpTpTpTpGpGpC), a heptanucleotide d(pCpCpApApApCpA), and Phn derivatives of the latter, it is shown that the introduction of a dye at the end of an oligonucleotide chain strongly stabilizes its complementary complexes. The Tmax and the thermodynamic parameters (delta H, delta S, delta G) of complex formation were determined. According to these data, coupling of a dye with the 5'-terminal phosphate group is the most advantageous: delta G(37 degrees C) is increased by 3.59 +/- 0.04 kcal/mol compared to 2.06 +/- 0.04 kcal/mol for 3'-Phn derivatives. The elongation of the linker, which connects the dye to the oligonucleotide, from a dimethylene up to a heptamethylene usually leads to destabilization of the oligonucleotide complex. The complementary complex formed by the 3',5'-di-Phn derivative of the heptanucleotide was found to be the most stable among all duplexes investigated. Relative to the unmodified complex the increase in free energy was 4.96 +/- 0.04 kcal/mol. The association constant of this modified complex at 37 degrees C is 9.5 x 10(6) M-1, whereas the analogous value for the unmodified complex is only 3 x 10(3) M-1.

[Site-directed chemical restriction of a single-stranded DNA fragment by an alkylating derivative of the tetranucleotide d(pApGpCpA) in the presence of tetranucleotide effectors]. / Saitnapravlennaia khimicheskaia restriktsiia odnotsepochechnogo fragmenta DNK alkiliruiushchim proizvodnym tetranukleotida d(pApGpCpA) v prisutstvii tetranukleotidnykh éffektorov.

Alkylation of a single-stranded DNA 302-mer by a 5'-O-phosphoryl-[4-(N-2-chloroethyl-N-methylamino)benzyl]amide derivative of the tetradeoxyribonucleotide d(pApGpCpA) in the presence of 3',5'-di-N-(2-hydroxyethyl) phenazinium derivatives of tetranucleotides as effectors led to specific chemical cleavage of the target at the guanosine residues of the sites ... pTpGppT. The reagent can be selectively addressed to one of three alkylation sites with the aid of a pair of tetranucleotide effectors flanking the chemically reactive tetranucleotide in the complex with the target DNA. The yield of the cleavage depends on the concentration of both the reagent and effectors, and can be enhanced, if a chain of two or more effectors from each side of the reagent is used. In this case, 3',5'-di-Phn-tetranucleotide effectors are to immediately flank the reagent.

[Effective selective modification of a single-stranded fragment of DNA with alkylating derivatives of short oligodeoxyribonucleotides in the presence of reaction effectors N-(2-hydroxyethyl)phenazine derivatives of oligodeoxyribonucleotides]. / Effektivnaia i selektivnaia modifikatsiia odnotsepochechnogo fragmenta DNK alkiliruiushchimi proizvodnymi korotkikh oligodezoksiribonukleotidov v prisutstvii éffektorov reaktsii N-(2-gidroksiétil)fenazinievykh proizvodnykh oligodezoksiribonukelotidov.

Tri-, tetra-, penta- and hexanucleotides bearing a reactive 4-(N-methylamino-N-2-chloroethyl)benzylamide group can effectively and selectively modify a single-stranded DNA fragment (302 nucleotides) in the presence of effectors, N-(2-hydroxyethyl)phenazinium derivatives of oligonucleotides complementary to DNA sequences adjacent to the binding site of the reagent. The reagents investigated modify not only single-stranded but also secondary-structured DNA regions. The modification extent depends on the length of oligonucleotide parts of the reagent and effector. A gap between the two stretches associated with the target DNA prevents the effector from functioning. The substitution of an octanucleotide effector by two tetranucleotide ones only slightly reduces the modification extent with a hexanucleotide reagent. A very efficient and specific modification can be achieved by using two effectors flanking the reactive oligonucleotide derivative. The approach leads to the modification extent of up to 89% with a hexanucleotide reagent.

N-(2-hydroxyethyl)phenazinium derivatives of oligonucleotides as effectors of the sequence-specific modification of nucleic acids with reactive oligonucleotide derivatives.

It has been found that mono- and especially diphenazinium derivatives of oligonucleotides complementary to the DNA sequence adjacent to the target sequence of the addressed alkylation of DNA, significantly enhance the extent and specificity of alkylation with p-(N-2-chloroethyl-N-methylamino)benzylamide derivatives of the addressing oligonucleotides, thus playing the role of effector of the sequence-specific (complementary addressed) modification.

[Modification of nucleic acids in stabilized complementary complexes. II. Effectiveness and direction of alkylation of dodecadeoxyribonucleotide d(pA-A-C-C-T-G-T-T-T-G-G-C) with 4-(N-2-chloroethyl-N-methylamino)benzylidene derivative of the complementary heptanucleotide d(pC-C-A-A-A-C)-A containing a N-(2-hydroxyethyl)phenazinium residue at the 5'-end]. / Modifikatsiia nukleinovykh kislot v stabilizirovannykh komplementarnykh kompleksakh. II. Effektivnost' i pozitsionnaia napravlennost' alkilirovaniia dodekadezoksiribonukleotida d(pA-A-C-C-T-G-T-T-T-G-G-C) 4-(N-khlorétil-N-metilamino) benzilidenovym proizvodnym komplementarnogo geptanukleotida d(pC-C-A-A-A-C)-A, soderzhashchim na 5'-kontse tsepi ostatok N-(2-oksiétil)fenaziniia.

Modification of dodecadeoxyribonucleotide d(pA-A-C-C-T-G-T-T-T-G-G-C) (I) with a heptanucleotide 4-(N-2-chloroethyl-N-methylamino)benzylidene (RCI) derivative d(pC-C-A-A-A-C) ARCI (II) and with similar reagent (III) bearing an additional 5'-terminal N-(2-hydroxyethyl)phenazinium residue (Phn) has been unvestigated. Both reagents (II) and (III) alkylated dodecanucleotide (I) mainly at the 5'-terminal phosphate, Phn residue not affecting specificity of the alkylation. Stabilization of the complementary complex target oligonucleotide-oligonucleotide derivative by the Phn group resulted in substantial increase of efficiency and rate of the intracomplex alkylation of the dodecanucleotide.

Complementary addressed modification and cleavage of a single stranded DNA fragment with alkylating oligonucleotide derivatives.

A single stranded DNA fragment was modified with alkylating derivatives of oligonucleotides complementary to a certain nucleotide sequences in the fragment. The derivatives carried aromatic 2-chloroethylamino groups at their 3'- or 5'-terminal nucleotide residues. Some of the derivatives carried both alkylating group and intercalating phenazine group which stabilized complementary complexes. It was found that these oligonucleotide derivatives modify the DNA fragment in a specific way near the target complementary nucleotide sequences, and the DNA fragment can be cleaved at the alkylated nucleotides positions. Alkylating derivatives carrying phenazine groups were found to be the most efficient in reaction with the DNA fragment.