DNA Aptamers against Taiwan Banded Krait α-Bungarotoxin Recognize Taiwan Cobra Cardiotoxins

Abstract

Bungarus multicinctus α-bungarotoxin (α-Bgt) and Naja atra cardiotoxins (CTXs) share a common structural scaffold, and their tertiary structures adopt three-fingered loop motifs. Four DNA aptamers against α-Bgt have been reported previously. Given that the binding of aptamers with targeted proteins depends on structural complementarity, in this study, we investigated whether DNA aptamers against α-Bgt could also recognize CTXs. It was found that N. atra cardiotoxin 3 (CTX3) reduced the electrophoretic mobility of aptamers against α-Bgt. Analysis of the changes in the fluorescence intensity of carboxyfluorescein-labeled aptamers upon binding toxin molecules revealed that CTX3 and α-Bgt could bind the tested aptamers. Moreover, the aptamers inhibited the membrane-damaging activity and cytotoxicity of CTX3. In addition to CTX3, other N. atra CTX isotoxins also bound to the aptamer against α-Bgt. Taken together, our data indicate that aptamers against α-Bgt show cross-reactivity with CTXs. The findings that aptamers against α-Bgt also suppress the biological activities of CTX3 highlight the potential utility of aptamers in regard to the broad inhibition of snake venom three-fingered proteins.

Keywords: aptamer; cardiotoxins; cytotoxicity; membrane-damaging activity; α-bungarotoxin.

Figure 1

Electrophoretic mobility shift assay of the binding of bgt1, bgt2, bgt3, and bgt4 aptamers to CTX3 and α-Bgt: 5 μM aptamers against α-Bgt was incubated with indicated concentrations of CTX3 and α-Bgt for 20 min, and then the aptamer-toxin mixtures were separated on 2% agarose gel.

Figure 2

Fluorescence spectra of FAM-bgt1-DABCYL and FAM-bgt1 in the presence of various CTX3 concentrations as indicated: (A) FAM-bgt1-DABCYL and (B) FAM-bgt1 in 10 mM Tris-HCl (pH 7.5) containing 1 mM EDTA and 100 mM NaCl were titrated with CTX3.

Figure 3

Effect of CTX3 and α-Bgt on fluorescence intensity of FAM-labeled bgt1, bgt2, bgt3, and bgt4 aptamers: the fluorescence intensity of FAM-labeled bgt1, bgt2, bgt3 and bgt4 was determined at 520 nm in the presence of various concentrations of (A) CTX3 or (B) α-Bgt. Fo and F represent the fluorescence intensity of FAM-labeled bgt1, bgt2, bgt3, and bgt4 in the absence and presence of toxins.

Figure 4

Effect of bgt1 (A), bgt2 (B), bgt3 (C), and bgt4 (D) aptamers on membrane-damaging activity of CTX3: CTX3 (0.23 μM) was incubated with 0.66, 1.65, and 3.30 μM aptamers against α-Bgt for 20 min, and then membrane-damaging activity of CTX3-aptamer mixtures were measured. Data represents mean ± SD of three independent experiments with triplicated measurements (* p < 0.05, vs. CTX3 without incubation with aptamers). The experiments were performed in 10 mM Tris-HCl (pH 7.5) containing 1 mM EDTA and 100 mM NaCl. The signal was expressed as the percentage of total calcein release after addition of 0.2% Triton X-100.

Figure 5

Effect of bgt1, bgt2, bgt3, and bgt4 aptamers on the cytotoxcity of CTX3: CTX3 (0.35 μM) was incubated with 1, 2, and 5 μM aptamers against α-Bgt for 20 min, and then the cytotoxicity of CTX3-aptamer mixtures were measured according to the procedure described in Materials and Methods section. Data represent mean ± SD of three independent experiments with triplicated measurements (* p < 0.05, vs. CTX3 without incubation with aptamers).

Figure 6

The fluorescence intensity of FAM-bgt1 in the presence of CTX isotoxins: (A) Fo and F represent the fluorescence intensity of FAM-bgt1 in the absence and presence of 1.5 μM CTXs. The fluorescence intensity was measured at 520 nm; and (B) amino acid sequence alignments of CTX1, CTX2, CTX3, CTX4, CTX5, CTXN, CLBP, and α-Bgt.