Synthesis and Characterization of Oligodeoxynucleotides Containing Naphthyridine:Imidazopyridopyrimidine Base Pairs at their Sticky Ends. Application as Thermally Stabilized Decoy Molecules

Abstract

We describe the synthesis and properties of oligodeoxynucleotides (ODNs) containing 1,8-naphthyridine C-nucleoside (Na-NO) and imidazo[5',4':4,5]pyrido[2,3-d]pyrimidine nucleoside (Im-ON) at the termini. The modified ODNs were more resistant (6 to 40 times) than natural DNA to snake venom phosphodiesterase (SVPD). Although incorporation of one pair each of Na-NO:Im-ON on the sticky ends of the duplex was insufficient for thermal stabilization (+2.5 degrees C per pair relative to the G:C pair), the duplex containing two consecutive Na-NO:Im-ON pairs at its sticky ends was markedly stabilized thermally. The stabilizing effect of the incorporation of additional Na-NO:Im-ON pairs is estimated to be +7.8 degrees C per pair. Application as thermally stabilized decoy molecules to NF-kappaB (p50) was also demonstrated. The DNA duplexes containing the Na-NO:Im-ON pairs (ODN I:ODN II and ODN III:ODN IV) acted as competitors to the natural NF-kappaB-binding duplex (ODN V: ODN VI), and the calculated IC50 values of ODN I:ODN II and ODN III:ODN IV were 20.1+/-13.3 and 10.9+/-4.8 nM, respectively, greater than that of ODN V:ODN VI.

Triazole-Linked Dumbbell Oligodeoxynucleotides with NF-κB Binding Ability as Potential Decoy Molecules

“…Preparation of Cross-Linking Precursor Oligodeoxynucleotides. Oligonucleotides containing N -3-(iodoethyl)thymidine at the 5‘-terminus and N -3-(propargylethyl)thymidine ( P ) at the 3‘-terminus were synthesized on a DNA synthesizer (Applied Biosystems Model 3400) in the DMTr-off mode, as we previously reported . CPG supporting ODNs was treated with 0.5 M NaN 3 in DMF (2 mL) at room temperature for 12 h. The CPG was filtrated, washed successively with ethanol and acetone, and dried.…”

“…Generally, relatively short double-stranded ODNs are used in the decoy strategy and possess less thermal stability under physiological conditions. Another drawback is the presence of extra- and intracellular nucleases, which cleave ODNs. , To achieve thermal stability and resistance to nucleases, structural alteration or chemical modification of decoy ODNs is necessary. − Dumbbell ODNs, which are circular ODNs consisting of double-stranded stem region and nucleotide loops at both of their termini, possess increased exonuclease resistance because they have no terminal nucleotide residues. − Dumbbell decoy ODNs against NF-κB were shown to inhibit in vitro and ex vivo transcription. These ODNs were prepared by enzymatically ligating two identical molecules. , However, in some cases, it is difficult to provide large quantities of modified ODNs by enzymatic preparation. , An alternative approach for cross-linking nucleic acids might be chemical modification, where a chemically reactive functional group is placed site-specifically in proximity to the opposing strands of a duplex .…”

Unnatural imidazopyridopyrimidine:naphthyridine base pairs: selective incorporation and extension reaction by Deep Vent (exo− ) DNA polymerase

“…This thermally stabilizing effect and specificity were considered to arise from (i) the complementary four H-bonds, (ii) the stacking ability of the expanded aromatic surface and (iii) the shape complementarity of the Im:Na base pairs similar to the purine:pyrimidine (pu:py) base pair. With these successful results in hand, we used the Im:Na base pairs to develop a thermally stabilized decoy molecule (9). Furthermore, we recently reported the enzymatic incorporation of dYTP (ImO N TP, NaN O TP, ImN O TP or NaO N TP) against the complementary base (NaN O , ImO N , NaO N or ImN O ) in the template by the Klenow fragment exo − [KF (exo − )] (10).…”

New imidazopyridopyrimidine:naphthyridine base-pairing motif, ImNN:NaOO, consisting of a DAAD:ADDA hydrogen bonding pattern, markedly stabilize DNA duplexes

“…+8 1C per pair relative to an A:T pair) independent of the sequence context, we attempted to develop thermally stabilized decoy molecules. 8 Furthermore, we have recently demonstrated the selective recognition of ImO N :NaN O and ImN O :NaO N pairs by DNA polymerases. 9,10 These successful results prompted us to develop new base-pairing motifs consisting of a series of Im:Na pairs.…”