New aspects of the interaction of the antibiotic coralyne with RNA: coralyne induces triple helix formation in poly(rA)*poly(rU)

Abstract

The interaction of coralyne with poly(A)*poly(U), poly(A)*2poly(U), poly(A) and poly(A)*poly(A) is analysed using spectrophotometric, spectrofluorometric, circular dichroism (CD), viscometric, stopped-flow and temperature-jump techniques. It is shown for the first time that coralyne induces disproportionation of poly(A)*poly(U) to triplex poly(A)*2poly(U) and single-stranded poly(A) under suitable values of the [dye]/[polymer] ratio (C(D)/C(P)). Kinetic, CD and spectrofluorometric experiments reveal that this process requires that coralyne (D) binds to duplex. The resulting complex (AUD) reacts with free duplex giving triplex (UAUD) and free poly(A); moreover, ligand exchange between duplex and triplex occurs. A reaction mechanism is proposed and the reaction parameters are evaluated. For C(D)/C(P)> 0.8 poly(A)*poly(U) does not disproportionate at 25 degrees C and dye intercalation into AU to give AUD is the only observed process. Melting experiments as well show that coralyne induces the duplex disproportionation. Effects of temperature, ionic strength and ethanol content are investigated. One concludes that triplex formation requires coralyne be only partially intercalated into AUD. Under suitable concentration conditions, this feature favours the interaction of free AU with AUD to give the AUDAU intermediate which evolves into triplex UAUD and single-stranded poly(A). Duplex poly(A)*poly(A) undergoes aggregation as well, but only at much higher polymer concentrations compared to poly(A)*poly(U).

Figure 1.

Coralyne chloride (8-methyl-2,3,10,11-tetramethoxydibenzo[a,g]quinolizinium chloride) molecular formula.

Figure 2.

Absorption spectra of the poly(A)•poly(U)/coralyne system. C_D = 4.9 × 10⁻⁵ M, C_P from 0 (a) to 1.3 × 10⁻³ M [C_D/C_P = 7.4 (b), 1.9 (c), 0.49 (d), 0.15 (e), 0.049 (f), 0.038 (g)], pH = 7.0, [NaCl] = 0.1 M, T = 25°C. As the RNA content is raised, the absorption first decreases and then increases, revealing that the reaction of dye binding to RNA is coupled to other processes.

Figure 3.

Fluorescence binding isotherms for different polynucleotide/coralyne systems. [NaCl] = 0.1 M, λ_ex = 420 nm, λ_em = 470 nm, T = 25°C. (A) poly(A)•poly(U), C_D = 7.6 × 10⁻⁷ M, pH = 7.0 (fit to Equation (4), the insert is an enlargement of the first part of the curve); (B) poly(A)•2poly(U), C_D = 7.2 × 10⁻⁷ M, pH = 7.0; (C) poly(A)•poly(A), C_D = 6.6 × 10 ^{− 7} M, pH = 5.2 (fit to Equation (6) of the first branch); (D) poly(A), C_D = 4.8 × 10⁻⁷ M, pH = 7.0. The biphasic behaviour displayed by poly(A)•poly(U) is also exhibited by the poly(A)•poly(A)/coralyne system, although to a limited extent. The usual form of the binding isotherm of poly(A)•2poly(U)/coralyne system reveals that in this case only dye binding to triplex is operative.

Figure 4.

Stern–Volmer plots for the NaI quenching of the light emitted by different polynucleotide/coralyne systems. C_D = 6.2 × 10 ^{− 7} M, λ_ex = 420 nm, λ_em = 470 nm, T = 25°C. (filled square) coralyne alone; (filled triangle) poly(A)•poly(U)/coralyne: C_P = 6.90 × 10⁻⁷ M, C_D/C_P = 0.9, %complex = 86, pH = 7.0, [NaCl] = 0.01 M; (open triangle) poly(A)•poly(U)/coralyne: C_P = 1.15 × 10⁻⁵ M, C_D/C_P = 0.05, %complex = 98, pH = 7.0, [NaCl] = 0.01 M; (filled circle) poly(A)•2poly(U)/coralyne: C_P = 1.15 × 10⁻⁵ M, C_D/C_P = 0.05, %complex = 99.6, pH = 7.0, [NaCl] = 0.1 M; (open circle) poly(A)•poly(A)/coralyne: C_P = 5.34 × 10⁻⁵ M, C_D/C_P = 0.01, %complex = 97, pH = 5.2, [NaCl] = 0.01 M. The maximum quenching effect is exerted on free coralyne in solution, whereas no quenching is exhibited by the poly(A)•2poly(U)/coralyne and poly(A)•poly(U)/coralyne at C_D/C_P = 0.05 systems because, owing to intercalation, coralyne is widely protected from the iodide action. The poly(A)poly(A) and poly(A)poly(U)/coralyne at C_D/C_P = 0.9 systems exhibit an intermediate behaviour, which suggests that in these systems coralyne is partially intercalated.

Figure 5.

Absorption melting profiles of the poly(A)•poly(U)/coralyne (columns A and B) and of the poly(A)•2poly(U)/coralyne systems (column C) at different C_D/C_P ratios. [NaCl] = 0.10 M, pH = 7.0, λ = 280 nm; (A) poly(A)•poly(U)/coralyne at C_D/C_P < 0.8, from top to bottom C_D/C_P = 0 (C_D = 0 M, C_P = 2.5 × 10⁻⁵ M), 0.1 (C_D = 1.7 × 10⁻⁵ M, C_P = 1.7 × 10 ^{− 4} M), 0.3 (C_D = 1.7 × 10⁻⁵ M, C_P = 5.7 × 10⁻⁵ M), 0.7 (C_D = 1.7 × 10⁻⁵ M, C_P = 2.7 × 10⁻⁵ M); (B) poly(A)•poly(U)/coralyne at C_D/C_P > 0.8, from top to bottom C_D/C_P = 0.9 (C_D = 1.7 × 10⁻⁵ M, C_P = 1.9 × 10⁻⁵ M), 1.0 (C_D = 1.7 × 10⁻⁵ M, C_P = 1.7 × 10⁻⁵ M), 2.0 (C_D = 1.7 × 10⁻⁵ M, C_P = 8.5 × 10⁻⁶ M), 3.0 (C_D = 1.7 × 10⁻⁵ M, C_P = 5.7 × 10⁻⁶ M); (C) poly(A)•2poly(U)/coralyne, from top to bottom C_D/C_P = 0 (C_D = 0 M, C_P = 5.0 × 10⁻⁴ M), 0.3 (C_D = 1.7 × 10⁻⁵ M, C_P = 5.7 × 10⁻⁵ M), 1.0 (C_D = 1.7 × 10⁻⁵ M, C_P = 1.7 × 10⁻⁵ M), 3.0 (C_D = 1.7 × 10⁻⁵ M, C_P = 5.7 × 10⁻⁶ M). For the poly(A)•poly(U)/coralyne system at 0 < C_D/C_P < 0.8 the prevailing form at low temperatures is the triplex; two transitions can be observed (triplex → duplex and then duplex → single-strands) that tend to merge when C_D/C_P approaches the value of 0.8. For C_D/C_P > 0.8 the absorbance decrease is related to the transition duplex → triplex, whereas the absorbance increase corresponds to the UAUD → U + A + U + D process.

Figure 6.

CD profile for the poly(A)•poly(U)/coralyne system. C_P = 6.2 × 10⁻⁵ M, C_D from 0 to 2.0 × 10 ^{− 4} M, [NaCl] = 0.10 M, pH = 7.0, T = 25°C, λ = 340 nm. The two inflexions can be observed in the CD profile. The first occurs at a value of C_D/C_P < 0.05 and the second at ∼0.9. The interval between the two inflections limits the range of C_D/C_P within which, at 25°C, poly(A)•poly(U) disproportionation do occur.

Figure 7.

Relative viscosity (η/η₀) dependence of poly(A)•poly(U)/coralyne (open triangle) and poly(A)•2poly(U)/coralyne (closed circle) systems on the C_D/C_P ratio. C_P = 1.6 × 10⁻⁴ M, C_D from 0 to 2.6 × 10 ^{− 4} M, [NaCl] = 0.10 M, pH = 7.0, T = 25°C. As expected, the two trends tend to overlap at low values of C_D/C_P because, under these circumstances, the polymer is present as a triplex in both systems. The trends become distinct at the highest values of C_D/C_P since the polymer is now present as a duplex in the poly(A)•poly(U)/coralyne system.

Figure 8.

Kinetic experiments showing triplex formation at [NaCl] = 0.10 M, pH = 7.0, T = 25°C, mixing time 5 ms. (A) Formation of the poly(A)•2poly(U)/coralyne complex is observed on mixing poly(A)•poly(U)/coralyne with poly(U); C_{poly(A)·poly(U)/coralyne} = 4.7 × 10⁻⁶ M, C_polyU = 4.7 × 10⁻⁶ M (the signal corresponds to the fluorescence change using λ_ex = 405 nm); (B) The experiment performed mixing poly(A)•poly(U) and coralyne at C_D/C_P = 3.0 (curve a) does not display any kinetic effect, thus indicating that triplex does not form under these conditions. In contrast, triplex formation is revealed by the absorbance decrease at 280 nm at C_D/C_P = 0.3 (curve b). Curve (c) shows that mixing the triplex poly(A)•2poly(U) with coralyne at C_D/C_P = 0.3 does not exhibit any signal change. (a) C_coralyne = 1.70 × 10⁻⁵ M; C_{poly(A)•poly(U)} = 5.67 × 10⁻⁶ M; (b) C_coralyne = 1.70 × 10⁻⁵ M; C_{poly(A)•poly(U)} = 5.67 × 10⁻⁵ M; (c) coralyne: C_coralyne = 1.70 × 10⁻⁵ M; C_{poly(A)•2poly(U)} = 5.67 × 10⁻⁵ M.

Figure 9.

T-jump experiments: dependence of the reciprocal relaxation time, 1/τ, on varying concentrations for the poly(A)•poly(U)/coralyne system at [NaCl] = 0.10 M, pH = 7.0, T = 25°C, λ_ex = 420 nm. (A) C_D from 3.4 × 10⁻⁷ M to 1.7 × 10 ^{− 6} M, C_P from 3.2 × 10⁻⁷ M to 4.9 × 10⁻⁶ M, C_D/C_P > 0.8, the binding of coralyne to duplex is observed [Step (1) of the reaction scheme], fit to Equation (8); (B) C_D from 1.1 × 10⁻⁶ M to 3.4 × 10⁻⁶ M, C_P from 2.6 × 10⁻⁵ M to 6.9 × 10⁻⁵ M, 0 < C_D/C_P < 0.8, dye exchange between duplex and triplex is observed [Step (3) of the reaction scheme], fit to Equation (9).