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

Abstract

New conjugates of triplex-forming pyrimidine oligo(2'-O-methylribonucleotides) with one or two 'head-to-head' hairpin oligo(N-methylpyrrole carboxamide) minor-groove binders (MGBs) attached to the terminal phosphate of the oligonucleotides with a oligo(ethylene glycol) linker were synthesized. It was demonstrated that, under appropriate conditions, the conjugates form stable complexes with double-stranded DNA (dsDNA) similarly to triplex-forming oligo(deoxyribonucleotide) (TFO) conjugates containing 5-methylated cytosines. Kinetic and thermodynamic parameters of the complex formation were evaluated by gel-shift assay and thermal denaturation. Higher melting temperatures (Tm), faster complex formation, and lower dissociation constants (Kd) of the triple helices (6-7 nM) were observed for complexes of MGB-oligo(2'-O-methylribonucleotide) conjugates with the target dsDNA compared to the nonconjugated individual components. Interaction of MGB moieties with the HIV proviral DNA fragment was indicated by UV/VIS absorption changes at 320 nm in the melting curves. The introduction of thymidine via a 3',3'-type 'inverted' phosphodiester linkage at the 3'-end of oligo(2'-O-methylribonucleotide) conjugates (3'-protection) had no strong influence on triplex formation, but slightly affected complex stability. At pH 6.0, when one or two hairpin MGBs were attached to the oligonucleotide, both triplex formation and minor-groove binding played important roles in complex formation. When two 'head-to-head' oligo(N-methylpyrrole) ligands were attached to the same terminal phosphate of the oligonucleotide or the linker, binding was observed at pH >7.5 and at high temperatures (up to 74 degrees). However, under these conditions, binding was retained only by the MGB part of the conjugate.

Triplex‐Forming Twisted Intercalating Nucleic Acids (TINAs): Design Rules, Stabilization of Antiparallel DNA Triplexes and Inhibition of G‐Quartet‐Dependent Self‐Association

“…When ONs 3 and 7 were incubated with target I, complete triplex formation www.chembiochem.org took over 5 h for both ONs, as detected using native PAGE ( Figure 5, see Figure 7SI and the Experimental Section for conditions applied). [6] TINA content does not seem to have a strong effect on this progress; however, a faster initiation of triplex formation was detected for ON 3 than for ON 7. To observe full conversion into the triplex, we recommend preincubation overnight.…”

“…It should be noted that in the case of parallel CT-triplexes, the unmodified triplex melts at 23 8C at pH 6. [6] 3) Of the TINA-TFOs tested, antiparallel triplexes are significantly more stable than parallel ones. Note that parallel TINATFOs (ONs 12 and 13) were designed in such a way that TINA positions were similar to the antiparallel TFO 7.…”

“…The binding affinities of TINATFOs were compared through their dissociation constants (K d ) from their triplexes (Table 1) with fluorescently-labeled target I ( Table 2). K d values were determined as described previously [6,34] using a non-denaturing gel-shift method in HEPES buffer at 37 8C, pH 7.2 (see Figure 1SI in the Supporting Information). Additionally, the intrinsic fluorescence of the TINA residues was followed in order to determine the aggregation state of the TINA oligonucleotides.…”

“…This buffer is most suitable for electrophoresis experiments at the indicated pH and it was also used in order to compare our results with those previously obtained. [6,34] The salt concentrations used were 50 mm NaCl, 150 mm NaCl, or 150 mm KCl. Glycerol containing bromophenol blue and xylene cyanol (up to 10 % of the glycerol concentration) was added to the samples (20 mL final volume), which were then heated at 90 8C for 3 min, slowly cooled down to room temperature and then incubated at +4 8C overnight.…”

“…TFO-MGB conjugates were obtained that could recognize DNA via the major and minor grooves. [5,6] MGB-MGB conjugates were also obtained that possess high affinity for their targets.…”