Cisplatin-induced duplex dissociation of complementary and destabilized short GG-containing duplex RNAs

Abstract

The ability of the anticancer active drug cisplatin to exert biological activity through interference with nucleic acid function is well documented. Since kinetics play a key role in determining product distributions in these systems, methods for accurate documentation of reactivity serve the purpose to identify preferential metal binding sites. In the present study, the aim has been to further explore a recently communicated approach (C. Polonyi and S. K. C. Elmroth, J. Chem. Soc., Dalton Trans., 2013, 42, 14959–14962) utilizing UV/vis spectroscopy and metal induced duplex RNA melting for monitoring of kinetics. More specifically, the sensitivity of the UV/vis-methodology has been evaluated by investigation of how overall length and changes of base-pairing in the close vicinity of a centrally located GG-site affect the rate of cisplatin binding, using the intracellularly active mono-aquated form of cisplatin (cis-Pt(NH₃)₂Cl(OH₂)⁺, (1a)) as the platination reagent. For this purpose, the reactivity of five different 13- to 17 base-pair duplex RNAs was monitored at 38 °C. A common trend of a ca. 10-fold reduction in reactivity was found to accompany an increase of bulk sodium concentration from C_Na+ = 122 mM to 1.0 M. Typical half-lives are exemplified by the interaction of 1a with the fully complementary 15-mer RNA-1 with t_1/2 = ca. 0.5 and 4.8 hours, at C_Na+ = 122 mM and 1.0 M respectively, and C_1a = 45 μM. Lowering of melting temperature (T_m) was found to promote reactivity regardless of whether the change involved a decrease or increase of the RNA length. For example, at C_Na+ = 1.0 M, truncation of the fully complementary and GG-containing 15-mer RNA-1 (T_m = 68.9 °C) to the 13-mer RNA-1-1-S (T_m = 63.9 °C) resulted in an increase of k_2,app from ca. 0.9 M⁻¹ s⁻¹ to 2.0 M⁻¹ s⁻¹_. Further, the 17-mer RNA-1-4 (T_m = 42.0 °C) with a central U₄ bulge exhibited the highest reactivity of the sequences studied with k_2,app = 4.0 M⁻¹ s⁻¹. The study shows that the reactivity of GG-sequences in RNA exhibit a strong variation depending on exact sequence context, and with imperfectly matched and/or stacked regions as particularly reactive sites.