Kinetics of cisplatin binding to short r(GG) containing miRNA mimics – influence of Na⁺versus K⁺, temperature and hydrophobicity on reactivity

Abstract

Nucleic acids are well recognized targets for platinum-based anticancer drugs, with RNA and DNA being kinetically comparable. In the case of RNA, previous studies have shown that the reaction between small duplex RNAs (dsRNAs) and monoaquated cisplatin (cis-Pt(NH3)2Cl(OH2)(+), ) can be followed by the metal induced hyperchromicity occurring directly after addition of to e.g. microRNA mimics. In the present study, we have used this approach to compare thermal stability and reactivity between intracellularly- and extracellularly relevant salt concentration (CNa(+) and CK(+)ca. 0.1 M), and also as a function of increased hydrophobicity (10% v/v EtOH). In addition, reactivity was studied as a function of temperature in the interval ca. 5-20 °C below the respective dsRNA melting temperatures (Tms). Four different 13- to 20-mer dsRNAs with two different central sequence motifs were used as targets containing either a central r(GG)·r(CC)- or r(GG)·r(UAU)-sequence. The reactions exhibited half-lives in the minute- to hour range at 38 °C in the presence of excess in the μM range. Further, a linear dependence was found between C and the observed pseudo-first-order rate constants. The resulting apparent second-order rate constants were significantly larger for the lower melting r(GG)·r(UAU)-containing sequences compared with that of the fully complementary ones; the higher and lower reactivities represented by RNA-1-3 and RNA-1-1 with k2,appca. 30 and 8 M(-1) s(-1) respectively at CNa(+) = 122 mM. For all RNAs a common small, but significant, trend was observed with increased reactivity in the presence of K(+) compared with Na(+), and decreased reactivity in the presence of EtOH. Finally, the temperature dependence of k2,app was evaluated using the Eyring equation. The retrieved activation parameters reveal positive values for both ΔH(≠) and ΔS(≠) for all dsRNAs, in the range ca. 23-34 kcal mol(-1) and 22-57 cal K(-1) mol(-1) respectively. These values indicate solvational effects to be important for the rate determining step of the reaction, and thus in support of a structural change of the dsRNA to take place in parallel with the adduct formation step.

Covalent and non-covalent binding of metal complexes to RNA

“…Moreover, they confirm that platination may be strongly affected by different parameters, including local RNA structure, cation nature and concentration, and hydrophobicity of the environment [45,70,79].…”

“…Interestingly, all tested duplexes show a small increase in reactivity in the presence of K(I), compared to Na(I), while ethanol seems to decrease the reactivity. These results suggest that the reaction between the mono-aquated form of cisplatin and short RNA duplexes is favoured in the intracellular rather than in the extracellular environment, but also show that the increased hydrophobicity of the environment reduces the reactivity [45].…”

“…In the sections below the most recent advances in the elucidation of platinum-RNA interactions are summarized, mainly focusing on examples subsequent to the above-mentioned review [39]. In particular, the following aspects are covered: (i) RNA platination kinetics [45,70,[77][78][79] (Sections 3.1 and 3.2); (ii) some examples of platinum binding to structured RNAs [80], focusing on latest insights into platinum binding to ribosomal RNA [44,76,81] (Section 3.3); (iii) the effect of platination on RNA interference [82][83][84] (Section 3.4); (iv) the use of Pt-RNA adducts to study RNA structure and dynamics [85] (Section 3.5) and (v) recent methods to detect RNA-platinum adducts in cells [47,86,87] (Section 3.6).…”

“…Surprisingly, the RNA binding abilities of small metal complexes, which can bind to RNA both covalently and non-covalently, have been much less explored [3,39,40]. However, a few recently published papers highlight the renewed interest of the scientific community for this topic [41][42][43][44][45][46][47].…”

(N7)-Platination and its effect on (N1)H-acidification in nucleoside phosphate derivatives

“…1 below) in microRNA mimics [5] regarding the binding kinetics of monoaquated Cisplatin, cis-[(NH 3 ) 2 PtCl(OH 2 )] + . The role of hydrogen transfer in redox processes involving nucleosides (i.e., the nucleobase and sugar residues) is yet another example [6].…”