Alternate Strand Triple Helix Formation Research Articles

Nucleosides and nucleotides. Part 226: Alternate-strand triple-helix formation by 3$prime;-3$prime;-linked oligodeoxynucleotides composed of asymmetrical sequences*1

Nucleosides and nucleotides. Part 226: Alternate-strand triple-helix formation by 3$prime;-3$prime;-linked oligodeoxynucleotides composed of asymmetrical sequences*1

Nucleosides and nucleotides. Part 226: Alternate-strand triple-helix formation by 3′-3′-linked oligodeoxynucleotides composed of asymmetrical sequences

In this paper, we describe the synthesis of the 3′-3′-linked oligonucleotides connected with pentaerythritol composed of asymmetrical sequences. Stability of the triplexes between these oligonucleotides and the DNA targets involving the adjacent oligopurine domains on alternate strands was investigated using the electrophoretic mobility shift assay (EMSA) and DNase I footprinting experiment. It was found that the 3′-3′-linked oligonucleotides composed of asymmetrical sequences formed the stable antiparallel triplexes with the DNA targets as compared with the unlinked oligonucleotides. Thus, oligonucleotides linked with pentaerythritol would be useful as antigene oligonucleotides for DNA targets consisting of the alternating oligopyrimidine–oligopurine sequences.

Oligonucleotides Containing an Acridine Group Covalently Bonded to the Nucleotide Flanking the 3′‐3′ Phosphodiester Junction for Alternate Strand Triple Helix Formation

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Oligonucleotides Containing an Acridine Group Covalently Bonded to the Nucleotide Flanking the 3′-3′ Phosphodiester Junction for Alternate Strand Triple Helix Formation

Oligonucleotides with a 3′-3′ inversion of polarity and containing an acridine group attached to nucleotide base flanking the 3′-3′ phosphodiester bon have been synthesized, characterized and used as third strand in alternate triple helix formation. CD melting studies and molecular mechanics calculations have been carried out to investigate these triplex structures.

Nucleosides and nucleotides. 218. Alternate-strand triple-helix formation by the 3'-3'-linked oligodeoxynucleotides using a purine motif.

In this paper, we describe the synthesis of the 3'-3'-linked TFOs that can form the antiparallel triplexes with the duplex DNA target by reverse Hoogsteen hydrogen bonds. Stability of the alternate-strand triplexes between these TFOs and the target DNAs was investigated using the electrophoretic mobility shift assay (EMSA). It was found that the alternate-strand triplexes were significantly stabilized by linking the TFO fragments with the pentaerythritol linker. And, unlike the alternate-strand triplexes composed of the pyrimidine motif, the terminal ammonium ion of the aminobutyl-linker and the intercalator of the TFOs did not contribute to the stability of the alternate-strand triplex comprised of the purine motif. We also tested the ability of the 3'-3'-linked TFOs to inhibit cleavage of the duplex DNA target 17 by the restriction enzyme EcoT14I and found that the 3'-3'-linked TFOs 12 and 13 inhibited the cleavage by the enzyme more effectively than the unlinked decamer 8. Thus, the TFOs linked with pentaerythritol may be useful as the antigene oligonucleotide to the DNA targets, which have alternating oligopyrimidine-oligopurine sequences.

Alternate-strand triple-helix formation by the 3'-3'-linked oligodeoxynucleotides with the intercalators at the junction point.

3'-3'-Linked oligodeoxynucleotides (ODNs) with the anthraquinonyl group at the junction point were synthesized on a DNA synthesizer using a controlled pore glass (CPG), which has pentaerythritol carrying the intercalator at one of the four hydroxymethyl groups. Stability of the triplexes with the target duplexes was studied by thermal denaturation. The 3'-3'-linked ODNs with the anthraquinonyl group enhanced the thermal stability of the triplexes when compared with those without the intercalator and the unmodified nonamer. The inhibitory activity of the 3'-3'-linked ODNs against the cleavage of the target DNA by the restriction enzyme Hind III was tested. It was found that the 3'-3'-linked ODN with the anthraquinonyl group at the junction point inhibited the cleavage by the enzyme more effectively than the nonamer and the 3'-3'-linked ODN without the intercalator.

Oligonucleotides containing a lysine residue as 3′–3′ junction for alternate strand triple helix formation

Oligonucleotides with a 3′–3′ inversion of polarity site assured by one lysine residue have been synthesized, characterized and used as third strands in alternate strand triple helix formation. UV melting studies and molecular mechanics calculations have been carried out to investigate the stability and the geometry of these new triplexes.

Nucleosides and nucleotides. 208. Alternate-strand triple-helix formation by the 3'-3'-linked oligodeoxynucleotides with the anthraquinonyl group at the junction point.

The synthesis of 3'-3'-linked oligodeoxynucleotides (ODNs) with the anthraquinonyl group at the junction point is described. The ODNs were synthesized on a DNA synthesizer using a controlled pore glass (CPG) carrying pentaerythritol that has an intercalator at one of the four hydroxymethyl groups. Stability of the triplexes with the target duplexes was studied by thermal denaturation. The 3'-3'-linked ODNs with the anthraquinonyl group enhanced the thermal stability of the triplexes when compared with those without the intercalator and the unmodified nonamer 10. It was found that the ODNs 12 and 13 carrying the anthraquinonyl groups can form thermally stable triplexes by skipping two or three extra base pairs between two binding domains of the target duplexes. The ability of the 3'-3'-linked ODNs to inhibit cleavage of the target DNA 22 by the restriction enzyme Hind III was tested. It was found that the 3'-3'-linked ODN 16 with the anthraquinonyl group at the junction point inhibited the cleavage by the enzyme more effectively than the nonamer 14 and the 3'-3'-linked ODN 15 without the intercalator.

Synthesis and characterization of new 3′-3′ linked oligodeoxyribonucleotides for alternate strand triple helix formation

Protected forms of 1,2,3-propanetriol and cis, cis-1,3,5-cyclohexanetriol were incorporated onto solid supports which were exploited in the solid phase synthesis of 3′-3′ linked oligodeoxyribonucleotides (ODNs), involving only nucleoside 3′-phosphoramidites as building blocks, UV thermal denaturation analysis showed the ability of ODNs with this inversion of polarity motif to cooperatively hybridize with duplexes of the type 5′-(Pu) m(Py) n-3′ in an alternate strand recognition approach. [Display omitted]

Optimization of alternate-strand triple helix formation at the 5"-TpA-3" and 5"-ApT-3" junctions.

Alternate-strand triple helix formation was optimized at the two junction steps, the 5"-TpA-3" and 5"-ApT-3" junctions. Footprint experiments, gel retardation assays and thermal denaturation measures on a sequence appropriately designed with two adjacent alternate-strand polypurine tracts points out that the addition of an adenine residue and the removal of one nucleotide should facilitate the crossing strands at the 5"-TpA-3" junction and at the 5"-ApT-3" junction, respectively. These results provide a 'switch code' for the construction of alternate-strand triple helix forming oligonucleotides which open new possibilities for extending the range of applications of antigene strategy.

New junction models for alternate-strand triple-helix formation.

[corrected] Oligonucleotide-directed triple-helix (triplex) formation can interfere with gene expression but only long tracts of oligopyrimidine*oligopurine sequences can be targeted. Attempts have been made to recognize short oligopurine sequences alternating on the two strands of double-stranded DNA by the covalent linkage of two triplex-forming oligonucleotides. Here we focus on the rational optimization of such an alternate-strand triplex formation on a DNA duplex containing a 5'-GpT-3'/3'-CpA-5' or a 5'-TpG-3'/3'-ApC-5' step by combination of (G,T)- and (G,A)-containing oligonucleotides that bind to the oligopurine strands in opposite orientations. The deletion of one nucleotide in the reverse Hoogsteen region of the oligonucleotide provides the best binding at the 5'GpT-3'/3'-CpA-5' step, whereas the addition of two cytosines as a linker between the two oligonucleotides is the best strategy to cross a 5'-TpG-3'/3'-ApC-5' step. Energy minimization and experimental data suggest that these two cytosines are involved in the formation of two novel base quadruplets. These data provide a rational basis for the design of oligonucleotides capable of binding to oligopurine sequences that alternate on the two strands of double-stranded DNA with a 5'-GpT-3'/3'-CpA-5' or a 5'-TpG-3'/3'-ApC-5' step at the junction.

Studies on Alternate Strand Triple Helix Formation by Oligodeoxyribonucleotides Containing A 3′-3′ Phosphodiester Bond

ODNs containing a 3′-3′ phosphodiester linkage as inversion of polarity motif have been shown to cooperatively bind to 5′-(purine)m(pyrimidine)n-3′ type duplexes by specific alternate strand recognition of the adjacent oligopurine domains. An NMR study has been undertaken to investigate the role of the 3′-3′ linked nucleosides and their nearest neighbours in the stabilization of the triple helical complexes.

DNA recognition by alternate strand triple helix formation: affinities of oligonucleotides for a site in the human p53 gene.

Duplex DNA recognition by oligonucleotide-directed triple helix formation is generally limited to homopurine target domains. Various approaches have been suggested for the relief of this constraint. Artificial DNA sequences have previously been used to show that adjacent homopurine domains on opposite DNA strands can be simultaneously recognized by oligonucleotide probes that switch triple helix recognition motifs between domains. Using assays of electrophoretic mobility and chemical protection, we have explored in detail whether such strategies are of benefit in designing high-affinity probes for a natural DNA sequence in the human p53 gene. This target site contains three adjacent, purine-rich domains on opposite DNA strands. Our results show that (i) a modest but statistically significant enhancement in affinity can be achieved for this sequence by designing an oligonucleotide that simultaneously recognizes all three purine domains, (ii) correction of a pyrimidine interruption in one purine domain does not dramatically alter this result, (iii) the relative energetic and structural contributions attributable to recognition of each purine domain can be assessed using probes with combinations of specific and nonspecific nucleotide sequences, and (iv) probe affinity is not correlated with the apparent number of base triplets for certain complexes. These data suggest that unfavorable free energy changes may be associated with alternation between triple helix motifs using existing strategies. In contrast to artificial DNA sequences optimized for this purpose, a substantial affinity enhancement was not observed using alternate strand DNA recognition at this natural target sequence. We therefore conclude that such enhancement is sequence dependent.

Recognition of double helical DNA by alternate strand triple helix formation

The triplet specificities and required strand orientations of two classes of DNA triple helices can be combined to target double helical sequences containing all four base pairs by alternate strand triple helix formation. This allows for the use of oligonucleotides containing only natural 3'-5' phosphodiester linkages to simultaneously bind both strands of double helical DNA in the major groove. The stabilities and structures of these alternate strand triple helices depend on whether the binding site sequence is 5'-(purine)_m(pyrimidine)_n-3' or 5'-(pyrimidine)_m(purine)_n-3'. The sequence type 5'-(purine)_m-(pyrimidine)_n-3' was targeted with an oligonucleotide consisting of a pyrimidine domain and a purine domain linked by a 3'-5' phosphodiester. To bind the duplex sequence type 5'-(pyrimidine)_m(purine)_n-3', the third strand requires at least two nucleotides linking binding domains at the site of crossover in the major groove. A new class of oligonucleotides capable of binding mixed duplex sequences by crossovers in the major groove may be possible.

Nonenymatic ligation of double-helical DNA by alternate-strand triple helix formation.

Nonenzymatic ligation of double-stranded DNA has been performed using an alternate-strand binding oligodeoxyribonucleotide template to juxtapose the duplex termini in a triple helical complex. The template associates with the duplex termini by Hoogsteen hydrogen bonding to alternate strands on opposite sides of the ligation site. Intermolecular and intramolecular ligation of linearized plasmid DNA are observed in the reaction, which depends on the template oligodeoxyribonucleotide and a condensing agent, N-cyanoimidazole. Intramolecular ligation products include those in which both strands are covalently closed in a circle. Ligation of the two strands is sequential and occurs at comparable rates for the first and second strands ligating. The covalent linkages formed in the reaction can be cleaved by the restriction endonuclease Stu I, supporting their identification as phosphodiesters.

Recognition of mixed-sequence duplex DNA by alternate-strand triple-helix formation

Oligodeoxyribonucleotide-directed triple-helix formation offers a chemical approach for the sequence-specific binding of double-helical DNA that is 10^6 times more specific than restriction enzymes. Because triple-helix formation by pyrimidine oligonucleotides is limited to purine tracts, it is desirable to find a general solution whereby oligonucleotides could be used to bind all four base pairs of intact duplex DNA (37 °C, pH 7.0). Approaches toward such a goal include the following: the search for other natural triplet specificities, such as G-TA triplets; the design of nonnatural bases for completion of the triplet code; the incorporation of abasic residues for nonreading of certain base pairs; and the design of oligonucleotides capable of binding alternate strands of duplex DNA by triple-helix formation. We report that a pyrimidine oligodeoxyribonucleotide-EDTA-Fe containing a 3'-3' phosphodiester and a 1,2-dideoxy-~ribose linker binds and cleaves a mixed-sequence double-helical DNA target site by alternate-strand triple-helix formation.