Oligonucleotide—Minor Groove Binder 1:2 Conjugates: Side by Side Parallel Minor Groove Binder Motif in Stabilization of DNA Duplex

Abstract

Synthetic polycarboxamides 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 when they form hairpin structures with side-by-side antiparallel motifs. In the present paper, new conjugates containing two ligands linked to the same terminal phosphate of DNA strand were constructed. The paper describes optimized synthesis and properties of oligonucleotide-linked polyamide strands that insert into the minor groove of a duplex in a parallel or antiparallel orientation. Strong stabilization of DNA duplexes by two attached minor groove ligands is demonstrated by the thermal denaturation method. The unmodified duplex 5'-CGTTTATTp-3'/5'-AATAAACG-3' melts at 20 degrees C. When one tetra(Py) residue was attached to the first strand of this duplex, denaturation temperature was increased to 46 degrees C; attachment of the second tetra(Py) in a parallel orientation resulted in denaturation temperature of 60 degrees C. It is even higher than in case of "classic" octapyrrole hairpin ligand (Tm = 58 degrees C). Sequence-specific character of stabilization by two conjugated ligands was demonstrated for G:C-containing oligonucleotides attached to tetracarboxamide and octacarboxamide ligands constructed from Py, Im and beta units according to established recognition rules (deltaTm = 20 degrees C). The two-strand parallel minor groove binder constructions attached to addressing oligonucleotides could be considered as site-specific ligands recognizing single- and double-stranded DNA similarly to already described hairpin MGB structures with antiparallel orientation of carboxamide units.

Sequence-Specific Conjugates of Oligo(2′-O-methylribonucleotides) and Hairpin Oligocarboxamide Minor-Groove Binders: Design, Synthesis, and Binding Studies with Double-Stranded DNA

“…In one of our previous publications [20], we have shown that a decrease in light absorption at 320 nm due to a shift in the absorption maximum during thermal denaturation experiments is a clear indication of the interaction and binding between dsDNA and oligocarboxamide MGBs. On the melting-curve derivatives at 320 nm, this effect is seen as a negative peak, with a minimum in a melting point of the dsDNA ± MGB complex.…”

Functionalized head-to-head hairpin polyamides: Synthesis, double-stranded DNA-binding activity and affinity

“…The advantage of the amide bonds in (7) and (9) is its stability at acid pH compared to phosphoramide bond in (3) and (6). 21 In addition, we noted that products (3) and (6) do not support heating up to 55°C and degrade in thermal denaturation experiment in contrast to the conjugates (7) and especially (9).…”

“…The other argument is that the natural HIV DNA fragment is the best target for molecule (9). It seems that the disposition of A/T and T/A plays an important role in bis-MGB binding and the binding blocks must be symmetrical relatively to central base pair, so that (T/A) 3-5 sequence must follow by (A/T) 3-5 sequence, as it is shown in Figure 5.…”

“…1). [7][8][9][10][11] To our surprise, the conjugates formed a strong complex with a target double-stranded DNA containing A/T-tracts even when the triplex could not exist (at neutral, slightly basic pH, and high temperatures). 10,11 Since under these conditions (close to physiological) the complex stability was provided only by MGB part of the conjugate, we decided to replace triplex-forming oligonucleotide (TFO) by a functional group in order to create a possibility for attachment of other molecules with .…”

“…Two hairpin hexa(N-methylpyrrolecarboxamides) H 2 Nc(Py) 3 c(Py) 3 Dp attached Ôhead-to headÕ to the terminal phosphate of a triplex-forming oligonucleotide (TFO), Dp, dimethylaminopropylamino group; c,c-aminobutyric acid residue, n = 0; 3 or 6. [9][10][11] DNA modifying activity. We joined N-termini of two standard hairpin hexa(N-methylpyrrole) carboxamides (each containing two blocks of 3 monomers connected by c-aminobutyric acid residue) in head-to-head orientation, similarly as shown in Figure 1.…”