Bulge oligonucleotide as an inhibitory agent of bacterial topoisomerase I

Abstract

Bacterial topoisomerase I (Btopo I) was defined as potential target for discovery of new antibacterial compounds. Various oligonucleotides containing bulge structure were designed and synthesised as inhibitors to Btopo I in this investigation. The results of this study demonstrated that the designed oligonucleotides display high inhibitory efficiency on the activity of Btopo I and the inhibitory effect could be modulated by the amount of bulge DNA bases. The most efficient one among them showed an IC₅₀ value of 63.1 nM in its inhibition on the activity of Btopo I. In addition, our studies confirmed that the designed oligonucleotide would induce irreversible damages to Btopo I and without any effects occur to eukaryotic topoisomerase I. It is our hope that the results provided in these studies could provide a novel way to inhibit Btopo I.

Keywords: Bacterial topoisomerase I; bulge oligonucleotide; inhibitor.

Figure 1.

Schematic representation of designed bulge oligonucleotide.

Figure 2.

Illustration of the supercoiled pUC 19 are catalysed by Btopo I in the presence or absence of bulge DNA.

Figure 3.

Determining the inhibitory effects to pUC 19 relaxation from B-1–1, B-1–2, B-1–3, B-1–4, B-1–5, and B-1–10. The concentration of bulge DNA was kept constant at 70 nM in all of the assay mixtures if added. The mixture containing 50 mM KAc, 20 mM Tris-Ac, 10 mM Mg(Ac)₂, 100 μg/ml BSA (pH 7.9 at 25° C), 250 ng pUC 19, 1 U of Btopo I, and the mixtures were incubated at 37 °C for 30 min before loading on agarose gel.

Figure 4.

pUC 19 relaxation assay for the inhibitory effects from B-1–1 (A), B-1–2 (B), B-1–3 (C), B-1–4 (D), B-1–5 (E), and B-1–10 (F). Assay mixture containing 50 mM KAc, 20 mM Tris-Ac (pH 8.0), 10 mM Mg(Ac)₂, 100 µg/ml BSA, 250 ng pUC 19, 1 U of Btopo I, and each bulge oligonucleotides were incubated at 37° C for 30 min before loading on agarose gel.

Figure 5.

Effects of reaction time on pUC 19 relaxation assay in the presence of B-1–10. The concentration of B-1–10 was kept constant at 200 nM in all of the assay mixtures if added. Assay mixtures containing 50 mM KAc, 20 mM Tris-Ac (pH 8.0), 10 mM Mg(Ac)₂, 100 µg/ml BSA, 250 ng pUC 19, 1 U of Btopo I, and each mismatch oligonucleotides were incubated at 37 °C for 0.5 h to 3.5 h from lane 2 to lane 6. In lane 7, additional 250 ng pUC 19 was added and 0.5 h reaction time was prolonged after the reaction reach 3.5 h.

Figure 6.

The relaxation assay was catalysed by Calf Topoisomerase I in the presence of different bulge DNA. The assay mixture containing 35 mM Tris-HCl (pH 8.0), 72 mM KCl, 5 mM MgCl₂, 5 mM DTT, 5 mM spermidine, 0.01% BSA, 250 ng pUC 19, 1 U calf Topoisomerase I, and each mixture was incubated at 37 °C for 30 min before loading on agarose gel.