Conjugation of a 3-(1H-phenanthro[9,10-d]imidazol-2-yl)-1H-indole intercalator to a triplex oligonucleotide and to a three-way junction.

A new intercalating nucleic acid monomer M comprising a 4-(1-indole)-butane-1,2-diol moiety was synthesized via a classical alkylation reaction of indole-3-carboxaldehyde followed by a condensation reaction with phenanthrene-9,10-dione in the presence of ammonium acetate to form a phenanthroimidazole moiety linked to the indole ring. Insertion of the new intercalator as a bulge into a Triplex Forming Oligonucleotide resulted in good thermal stability of the corresponding Hoogsteen-type triplexes. Molecular modeling supports the possible intercalating ability of M. Hybridisation properties of DNA/DNA and RNA/DNA three-way junctions (TWJ) with M in the branching point were also evaluated by their thermal stability at pH 7. DNA/DNA TWJ showed increase in thermal stability compared to wild type oligonucleotides whereas this was not the case for RNA/DNA TWJ.

Synthesis of locked pyranosyl nucleic acid (LpNA).

A new locked pyranosyl nucleoside was synthesized by phenylsulfinyl-assisted chemistry. The novel building block was inserted into oligonucleotides and provides new insight on conformational restricted pyranosyl nucleosides on duplex formation.

Cationic modified nucleic acids for use in DNA hairpins and parallel triplexes.

Non-nucleosidic DNA monomers comprising partially protonated amines at low pH have been designed and synthesized. The modifications were incorporated into DNA oligonucleotides via standard DNA phosphoramidite synthesis. The ability of cationic modifications to stabilize palindromic DNA hairpins and parallel triplexes were evaluated using gel electrophoresis, circular dichroism and thermal denaturation measurements. The non-nucleosidic modifications were found to increase the thermal stability of palindromic hairpins at pH 8.0 as compared with a nucleosidic tetraloop (TCTC). Incorporation of modifications at the 5'-end of a triplex forming oligonucleotide resulted in a significant increase in thermal stability at low pH when the modifications were placed as the 5'-dangling end.

Using an aryl phenanthroimidazole moiety as a conjugated flexible intercalator to improve the hybridization efficiency of a triplex-forming oligonucleotide.

When inserting 2-phenyl or 2-naphth-1-yl-phenanthroimidazole intercalators (X and Y, respectively) as bulges into triplex-forming oligonucleotides, both intercalators show extraordinary high thermal stability of the corresponding Hoogsteen-type triplexes and Hoogsteen-type parallel duplexes with high discrimination to Hoogsteen mismatches. Molecular modeling shows that the phenyl or the naphthyl ring stacks with the nucleobases in the TFO, while the phenanthroimidazol moiety stacks with the base pairs of the dsDNA. DNA-strands containing the intercalator X show higher thermal triplex stability than DNA-strands containing the intercalator Y. The difference can be explained by a lower degree of planarity of the intercalator in the case of naphthyl. It was also observed that triplex stability was considerably reduced when the intercalators X or Y was replaced by 2-(naphthlen-1-yl)imidazole. This confirms intercalation as the important factor for triplex stabilization and it rules out an alternative complexation of protonated imidazole with two phosphate groups. The intercalating nucleic acid monomers X and Y were obtained via a condensation reaction of 9,10-phenanthrenequinone (4) with (S)-4-(2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethoxy)benzaldehyde (3a) or (S)-4-(2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethoxy)-1-naphthaldehyde (3b), respectively, in the presence of acetic acid and ammonium acetate. The required monomers for DNA synthesis using amidite chemistry were obtained by standard deprotection of the hydroxy groups followed by 4,4'-dimethoxytritylation and phosphitylation.

High physiological thermal triplex stability optimization of twisted intercalating nucleic acids (TINA).

The structure of the monomer (R)-1-O-[4-(1-pyrenylethynyl)phenylmethyl]glycerol () in twisted intercalating nucleic acids (TINA) was optimized for stabilizing interactions between the intercalator and surrounding nucleobases when used as a triplex forming oligonucleotide (TFO). Enhancement of pi-pi interactions with nucleobases of the TFO was achieved by increasing the aromatic surface using the (R)-1-O-[4-(1-pyrenylethynyl)naphthylmethyl]glycerol monomer (). Bulge insertion of in the middle of a Hoogsteen-type triplex increased the triplex thermal stability, DeltaT(m) = +2.0 degrees C compared with at pH 7.2. Syntheses and thermal denaturation studies of triplexes and duplexes are described for three novel TINA monomers. The influence of pi-pi interactions, link length and the positioning of the ether in the linker in the TINA derivatives are described.

Triplex glue by synthesizing conjugated flexible intercalators.

Bulge insertions of conjugated intercalators into the DNA triplex structure are found to give a dramatic contribution to the triplex stability. On the other hand insertions of conjugated intercalators are found to diminish quadruplex structures and in this way breaking down the self association of G-rich oligonucleotides under physiologically potassium ion conditions. A large number of intercalators are described here and they all result in dramatic increases of thermal stability of the corresponding triplexes. Another interesting aspect of conjugated intercalators is their use for assembling alternate strand triplexes. Targeting of neighbouring purine sequences on each their strand in the duplex DNA is a challenge for the 5'- 5' connectivity of the TFOs because of a large distance between the 5'-ends. The intercalator approach offers a linkage with the proper combination of flexibility and rigidity to produce alternate strand triplexes with higher stability than a similar wild type triplex of the same total length.

Purine twisted-intercalating nucleic acids: a new class of anti-gene molecules resistant to potassium-induced aggregation.

Sequence-specific targeting of genomic DNA by triplex-forming oligonucleotides (TFOs) is a promising strategy to modulate in vivo gene expression. Triplex formation involving G-rich oligonucleotides as third strand is, however, strongly inhibited by potassium-induced TFO self-association into G-quartet structures. We report here that G-rich TFOs with bulge insertions of (R)-1-O-[4-(1-pyrenylethynyl)-phenylmethyl] glycerol (called twisted intercalating nucleic acids, TINA) show a much lower tendency to aggregate in potassium than wild-type analogues do. We designed purine-motif TINA-TFOs for binding to a regulatory polypurine-polypyrimidine (pur/pyr) motif present in the promoter of the KRAS proto-oncogene. The binding of TINA-TFOs to the KRAS target has been analysed by electrophoresis mobility shift assays and DNase I footprinting experiments. We discovered that in the presence of potassium the wild-type TFOs did not bind to the KRAS target, differently from the TINA analogues, whose binding was observed up to 140 mM KCl. The designed TINA-TFOs were found to abrogate the formation of a DNA-protein complex at the pur/pyr site and to down-regulate the transcription of CAT driven by the murine KRAS promoter. Molecular modelling of the DNA/TINA-TFO triplexes are also reported. This study provides a new and promising approach to create TFOs to target in vivo the genome.

Triplex formation by pyrene-labelled probes for nucleic acid detection in fluorescence assays.

Triplex-forming homopyrimidine oligonucleotides containing insertions of a 2'-5' uridine linkage featuring a pyrene moiety at the 3'-position exhibit strong fluorescence enhancement upon binding to double-stranded DNA through Hoogsteen base pairing. It is shown that perfect matching of the new modification to the base pair in the duplex is a prerequisite for strong fluorescence, thus offering the potential to detect single mutations in purine stretches of duplex DNA. The increase in the fluorescence signal was dependent on the thermal stability of the parallel triplex, so a reduction in the pH from 6.0 to 5.0 resulted in an increase in thermal stability from 25.0 to 55.0 degrees C and in an increase in the fluorescence quantum yield (Phi(F)) from 0.061 to 0.179, while the probe alone was fluorescently silent (Phi(F)=0.001-0.004). To achieve higher triplex stability, five nucleobases in a 14-mer sequence were substituted with alpha-L-LNA monomers, which provided a triplex with a T(m) of 49.5 degrees C and a Phi(F) of 0.158 at pH 6.0. Under similar conditions, a Watson-Crick-type duplex formed with the latter probe showed lower fluorescence intensity (Phi(F)=0.081) than for the triplex.