Sequence-specific recognition of double-stranded DNA by synthetic minor groove binder conjugates. toward the construction of artificial site-specific deoxyribonucleases

Bis-conjugates of hairpin N-methylpyrrole/N-methylimidazole oligocarboxamide minor groove binders (MGB) possessing enhanced affinity and sequence-specificity for dsDNA were synthesized. Two hairpin MGBs were connected by their N-termini via an aminodiacetate linker. The binding of bis-MGB conjugates to the target DNA was studied by gel mobility retardation, footprinting, and circular dichroism; their affinity and binding mode in the DNA minor groove were determined. In order to functionalize the bis-MGB conjugates, DNA-cleaving agents such as phenanthroline or bipyridine were attached. Effective site-specific cleavage of target DNA in the presence of Cu(2+) ions was observed.     

Head-to-head bis-hairpin polyamide minor groove binders and their conjugates with triplex-forming oligonucleotides: studies of interaction with target double-stranded DNA

Two hairpin hexa(N-methylpyrrole)carboxamide DNA minor groove binders (MGB) were linked together via their N-termini in head-to-head orientation. Complex formation between these bis-MGB conjugates and target DNA has been studied using DNase I footprinting, circular dichroism, thermal dissociation, and molecular modeling. DNase I footprint revealed binding of these conjugates to all the sites of 492 b.p. DNA fragment containing (A/T)(n)X(m)(A/T)(p) sequences, where n>3, p>3; m=1,2; X = A,T,G, or C. Binding affinity depended on the sequence context of the target. CD experiments and molecular modeling showed that oligo(N-methylpyrrole)carboxamide moieties in the complex form two short antiparallel hairpins rather than a long parallel head-to-head hairpin. Binding of bis-MGB also stabilized a target duplex thermodynamically. Sequence specificity of bis-MGB/DNA binding was validated using bis-conjugates of sequence-specific hairpin (N-methylpyrrole)/(N-methylimidazole) carboxamides. In order to increase the size of recognition sequence, the conjugates of bis-MGB with triplex-forming oligonucleotides (TFO) were synthesized and compared to TFO conjugated with single MGB hairpin unit. Bis-MGB-oligonucleotide conjugates also bind to two blocks of three and more A.T/T.A pairs similarly to bis-MGB alone, independently of the oligonucleotide moiety, but with lower affinity. However, the role of TFO in DNA recognition was demonstrated for mono-MGB-TFO conjugate where the binding was detected mainly in the area of the target sequence consisting of both MGB and TFO recognition sites. Basing on the molecular modeling, three-dimensional models of both target DNA/bis-MGB and target DNA/TFO-bis-MGB complexes were built, where bis-MGB forms two antiparallel hairpins. According to the second model, one MGB hairpin is in the minor groove of 5'-adjacent A/T sequence next to the triplex-forming region, whereas the other one occupies the minor groove of the TFO binding polypurine tract. All these data together give a key information for the construction of MGB-MGB and MGB-oligonucleotide conjugates possessing high specificity and affinity for the target double-stranded DNA.     

Stabilization of DNA double and triple helices by conjugation of minor groove binders to oligonucleotides

New conjugates containing two parallel or antiparallel carboxamide minor groove binders (MGB) attached to the same terminal phosphate of one oligonucleotide strand were synthesized. The conjugates interact with their target DNA stronger than the individual components. Effect of conjugated MGB on DNA duplex and triplex stability and their sequence specificity was demonstrated on the short oligonucleotide duplexes and on the triplex formed by model 16-mer oligonucleotide with HIV polypurine tract.     

Stabilization of DNA Double and Triple Helices by Conjugation of Minor Groove Binders to Oligonucleotides

Abstract

New conjugates containing two parallel or antiparallel carboxamide minor groove binders (MGB) attached to the same terminal phosphate of one oligonucleotide strand were synthesized. The conjugates interact with their target DNA stronger than the individual components. Effect of conjugated MGB on DNA duplex and triplex stability and their sequence specificity was demonstrated on the short oligonucleotide duplexes and on the triplex formed by model 16-mer oligonucleotide with HIV polypurine tract.     

Conjugates of oligo(2'-O-methylribonucleotides) with minor groove binders as new sequence-specific agents recognizing both grooves of double-stranded DNA

Design, synthesis and physico-chemical studies of new pyrimidine oligo(2'-O-methylribonucleotide) conjugates with one or two oligo(pyrrolecarboxamide) minor groove binders (MGB) are described.     

Oligonucleotide-minor groove binder conjugates and their complexes with complementary DNA: effect of conjugate structural factors on the thermal stability of duplexes

Synthetic polycarboxamide minor groove binders (MGB) consisting of N-methylpyrrole (Py), N-methylimidazole (Im), N-methyl-3-hydroxypyrrole (Hp) and beta-alanine (beta) show strong and sequence-specific interaction with the DNA minor groove in side-by-side antiparallel or parallel orientation. Two MGB moieties covalently linked to the same terminal phosphate of one DNA strand stabilize DNA duplexes formed by this strand with a complementary one in a sequence-specific manner, similarly to the corresponding mono-conjugated hairpin structures. The series of conjugates with the general formula Oligo-(L-MGB-R)m was synthesized, where m = 1 or 2, L = linker, R = terminal charged or neutral group, MGB = -(Py)n-, -(Im)n- or -[(Py/Im)n-(CH2)3CONH-(Py/Im)n-] and I < n < 5. Using thermal denaturation, we studied effects of structural factors such as m and n, linker L length, nature and orientation of the MGB monomers, the group R and the backbone (DNA or RNA), etc. on the stability of the duplexes. Structural factors are more important for linear and hairpin monophosphoroamidates than for parallel bis-phosphoroamidates. No more than two oligocarboxamide strands can be inserted into the duplex minor groove. Attachment of the second sequence-specific parallel ligand [-L(Py)4R] to monophosphoroamidate conjugate CGTTTATT-L(Py)4R leads to the increase of the duplex Tm, whereas attachment of [-L(Im)4R] leads to its decrease. The mode of interaction between oligonucleotide duplex and attached ligands could be different (stacking with the terminal A:T pair of the duplex or its insertion into the minor groove) depending on the length and structure of the MGB.     

Synthesis and Binding Properties of Oligonucleotides Containing an Azobenzene Linker

Abstract

Incorporation of an azobenzene-4,4′-diamide group via a linker arm into the 3′-hydroxyl function of one oligonucleotide segment and the 5′-OH of other oligonucleotide has been described. The binding of the oligonucleotides containing the azobenzene linker was investigated by UV melting behaviors. The azobenzene linker has been shown to be useful as an effective bridge for stabilizing hairpin duplex and triplex.     

Oligonucleotide conjugates with minor groove ligands as probes for hybridization microarray chips

A possibility of using oligonucleotide conjugates with minor groove ligands as probes for hybridization microarray chips was studied. The oligonucleotide conjugates contain a hairpin ligand (MGB) composed of two tripyrrolcarboxamide residues with an aminocaproic acid residue as a linker and bound to the oligonucleotide duplex AT tract in a site-specific manner. We used as (5'-3') probes GACAAGAp, GACAAAAp, GACAAGA-MGB, and GACAAAA-MGB. The oligonucleotides labeled with Cy3 cyanine dye, Cy3-ACTAATTTTGTC and Cy3-ACTAATCTTGTC, were used as targets. The maximal MGB effect on the fluorescence level of microarray chip spots, which caused its fourfold increase as compared with the initial unmodified duplex, was observed for the duplex containing only AT pairs in the ligand binding site. The presence of A-C and G-T mutations in the binding site (imperfect duplexes) or a C-G pair (perfect duplex) affects the change in fluorescence level to a considerably lesser degree.     

Oligonucleotide conjugates with minor groove ligands as probes for hybridization microarray chips

A possibility of using oligonucleotide conjugates with minor groove ligands as probes for hybridization microarray chips was studied. The oligonucleotide conjugates contain a hairpin ligand (MGB) composed of two tripyrrolcarboxamide residues with an aminocaproic acid residue as a linker and bound to the oligonucleotide duplex AT tract in a site-specific manner. We used as (5′-3′)-probes: GACAAGAp, GACAAAAp, GACAAGA-MGB, and GACAAAA-MGB. The oligonucleotides labeled with the Cy3 cyanine dye, Cy3-ACTAATTTTGTC and Cy3-ACTAATCTTGTC, were used as targets. The maximal MGB effect on the fluorescence level of microarray chip spots, which caused its fourfold increase as compared with the initial unmodified duplex, was observed for the duplex containing only AT pairs in the ligand binding site. The presence of AC and GT mutations in the binding site (imperfect duplexes) or a CG pair (perfect duplex) affect the change in fluorescence level to a considerably lesser degree.     

Triple helix-forming oligonucleotides conjugated to indolocarbazole poisons direct topoisomerase I-mediated DNA cleavage to a specific site.

Topoisomerase I is an ubiquitous DNA-cleaving enzyme and an important therapeutic target in cancer chemotherapy for camptothecins as well as for indolocarbazole antibiotics such as rebeccamycin. To achieve a sequence-specific cleavage of DNA by topoisomerase I, a triple helix-forming oligonucleotide was covalently linked to indolocarbazole-type topoisomerase I poisons. The three indolocarbazole-oligonucleotide conjugates investigated were able to direct topoisomerase I cleavage at a specific site based upon sequence recognition by triplex formation. The efficacy of topoisomerase I-mediated DNA cleavage depends markedly on the intrinsic potency of the drug. We show that DNA cleavage depends also upon the length of the linker arm between the triplex-forming oligonucleotide and the drug. Based on a known structure of the DNA-topoisomerase I complex, a molecular model of the oligonucleotide conjugates bound to the DNA-topoisomerase I complex was elaborated to facilitate the design of a potent topoisomerase I inhibitor-oligonucleotide conjugate with an optimized linker between the two moieties. The resulting oligonucleotide-indolocarbazole conjugate at 10 nM induced cleavage at the triple helix site 2-fold more efficiently than 5 microM of free indolocarbazole, while the other drug-sensitive sites were not cleaved. The rational design of drug-oligonucleotide conjugates carrying a DNA topoisomerase poison may be exploited to improve the efficacy and selectivity of chemotherapeutic cancer treatments by targeting specific genes and reducing drug toxicity.     

Spectroscopic studies on the formation and thermal stability of DNA triplexes with a benzoannulated delta-carboline-oligonucleotide conjugate

A benzoannulated delta-carboline with a phenyl substituent has been covalently tethered to the 3'-end of a triplex-forming oligonucleotide and its ability to bind and stabilize DNA triple helices has been examined by various spectroscopic methods. UV thermal melting experiments were conducted with different hairpin duplexes and with a complementary single-stranded oligonucleotide as targets for the conjugate. The delta-carboline ligand preferentially binds triplexes over duplexes and leads to a temperature increase of the triplex-to-duplex transition by up to 23 degrees C. The results obtained from UV, CD and fluorescence measurements suggest that the delta-carboline ligand exhibits specific interactions with a triplex and favors binding by intercalation at the triplex-duplex junction.     

Strand displacement of double-stranded DNA by triplex-forming antiparallel purine-hairpins

We characterize the binding affinity and the thermodynamics of hybridization of triplex-forming antiparallel purine-hairpins composed of two antiparallel purine domains linked by a loop directed toward single-stranded and double-stranded DNA (ssDNA, dsDNA). Gel retardation assays and melting experiments reveal that a 13-mer purine-hairpin binds specifically and with a K ( d ) of 8 x 10(8) M to polypyrimidine ssDNA to form a triple helical structure. Remarkably, we show that purine-hairpins also bind polypurine/polypyrimidine stretches included in a dsDNA of several hundred bp in length. Binding of purine-hairpins to dsDNA occurs by triplex formation with the polypyrimidine strand, causing displacement of the polypurine strand. Because triplex formation is restricted to polypurine/polypyrimidine stretches of dsDNA, we studied the triplex formation between purine-hairpins and polypyrimidine targets containing purine interruptions. We found that an 11-mer purine-hairpin with an adenine opposite to a guanine interruption in the polypyrimidine track binds to ssDNA and dsDNA, allowing expansion of the possible target sites and increase in the length of purine-hairpins. Thus, when using a 20-mer purine-hairpin targeting an interruption-containing polypyrimidine target, the binding affinity is increased compared to its 13-mer antiparallel purine-hairpin counterpart. Surprisingly, this increase is much more pronounced than that observed for a tail-clamp purine-hairpin extended up to 20 nt in the Watson-Crick domain only. Thus, triplexforming antiparallel purine-hairpins can be a potentially useful strategy for both single-strand and double-strand nucleic acid recognition.     

Secondary binding sites for triplex-forming oligonucleotides containing bulges, loops, and mismatches in the third strand

We have used DNase I footprinting to examine the binding of five different 17-mer oligonucleotides to a 53-base oligopurine tract containing four pyrimidine interruptions. Although all the expected triplexes formed with high affinity (K(d) approximately 10-50 nM), one oligonucleotide produced a footprint at a second site with about 20-fold lower affinity. We have explored the nature of this secondary binding site and suggest that it arises when each end of the third strand forms a 7-mer triplex with adjacent regions on the duplex, generating a contiguous 14-base triplex with a bulge in the center of the third strand oligonucleotide. This unusual binding mode was examined by use of oligonucleotides that were designed with the potential to form different length third-strand loops of various base composition. We find that triplexes containing single-base bulges are generally more stable than those with dinucleotide loops, though triplexes can be formed with loops of up to nine thymines, generating complexes with submicromolar dissociation constants. These structures are much more stable than those formed by adding two separate 7-mer oligonucleotides, which do not generate DNase I footprints, though a stable complex is generated when the two halves are covalently joined by a hexa(ethylene glycol) linker. MPE produces less clear footprints, presumably because this cleavage agent binds to triplex DNA, but confirms that the oligonucleotides can bind in unexpected places. These results suggest that extra care needs to be taken when designing long triplex-forming oligonucleotides so as to avoid triplex formation at shorter secondary sites.     

Triple helix formation with purine-rich phosphorothioate-containing oligonucleotides covalently linked to an acridine derivative.

Purine-rich (GA)- and (GT)-containing oligophosphorothioates were investigated for their triplex-forming potential on a 23 bp DNA duplex target. In our system, GA-containing oligophosphorothioates (23mer GA-PS) were capable of triplex formation with binding affinities lower than (GA)-containing oligophosphodiesters (23mer GA-PO). The orientation of the third strand 23mers GA-PS and GA-PO was antiparallel to the purine strand of the duplex DNA target. In contrast, (GT)-containing oligophosphorothioates (23mer GT-PS) did not support triplex formation in either orientation, whereas the 23mer GT-PO oligophosphodiester demonstrated triplex formation in the antiparallel orientation. GA-PS oligonucleotides, in contrast to GT-PS oligonucleotides, were capable of self-association, but these self-associated structures exhibited lower stabilities than those formed with GA-PO oligonucleotides, suggesting that homoduplex formation (previously described for the 23mer GA-PO sequence by Noonberg et al.) could not fully account for the decrease in triplex stability when phosphorothioate linkages were used. The 23mer GA-PS oligonucleotide was covalently linked via its 5'-end to an acridine derivative (23mer Acr-GA-PS). In the presence of potassium cations, this conjugate demonstrated triplex formation with higher binding affinity than the unmodified 23mer GA-PS oligonucleotide and even than the 23mer GA-PO oligonucleotide. A (GA)-containing oligophosphodiester with two phosphorothioate linkages at both the 5'- and 3'-ends exhibited similar binding affinity to duplex DNA compared with the unmodified GA-PO oligophosphodiester. This capped oligonucleotide was more resistant to nucleases than the GA-PO oligomer and thus represents a good alternative for ex vivo applications of (GA)-containing, triplex-forming oligonucleotides, allowing a higher binding affinity for its duplex target without rapid cellular degradation.     

Efficient cleavage of DS DNA by bleomycin conjugated via hexaethylene glycol linker to triplex-forming oligonucleotides

New conjugates of bleomycin A5 with oligonucleotides are synthesized. The bleomycin residue was attached to the 3'- or 5'- terminus of hexadecathymidilate via a hexaethylene glycol phosphate linker. Newly designed conjugates were shown to cleave site-specifically both strands of a dsDNA fragment within a triplex. The maximum extent of cleavage for individual strand amounts to 61%.     

Promotion of triplex formation by 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) modification: thermodynamic and kinetic studies

We analyzed the effect of 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) modification of triplex-forming oligonucleotide (TFO) on pyrimidine motif triplex formation at neutral pH, a condition where pyrimidine motif triplexes are unstable. The binding constant of the pyrimidine motif triplex formation at pH 6.8 with 2',4'-BNA modified TFO was about 20 times larger than that observed with unmodified TFO. The observed increase in the binding constant at neutral pH by the 2',4'-BNA modification resulted from the considerable decrease in the dissociation rate constant.