Non-identical electronic characters of the internucleotidic phosphates in RNA modulate the chemical reactivity of the phosphodiester bonds

Abstract

We here show that the electronic properties and the chemical reactivities of the internucleotidic phosphates in the heptameric ssRNAs are dissimilar in a sequence-specific manner because of their non-identical microenvironments, in contrast with the corresponding isosequential ssDNAs. This has been evidenced by monitoring the δH8() shifts upon pH-dependent ionization (pK_a1) of the central 9-guaninyl () to the 9-guanylate ion (G⁻), and its electrostatic effect on each of the internucleotidic phosphate anions, as measured from the resultant δ³¹P shifts (pK_a2) in the isosequential heptameric ssRNAs vis-à-vis ssDNAs: [d/r(5′-Cp₁Ap₂Q¹p₃p₄Q²p₅Ap₆C-3′): Q¹ = Q² = A (5a/5b) or C (8a/8b), Q¹ = A, Q² = C (6a/6b), Q¹ = C, Q² = A (7a/7b)]. These oligos with single ionizable in the centre are chosen because of the fact that the pseudoaromatic character of can be easily modulated in a pH-dependent manner by its transformation to G⁻ (the 2′-OH to 2-O⁻ ionization effect is not detectable below pH 11.6 as evident from the N^1-Me- analog), thereby modulating/titrating the nature of the electrostatic interactions of to G⁻ with the phosphates, which therefore constitute simple models to interrogate how the variable pseudoaromatic characters of nucleobases under different sequence context (J. Am. Chem. Soc., 2004, 126, 8674–8681) can actually influence the reactivity of the internucleotide phosphates as a result of modulation of sequence context-specific electrostatic interactions. In order to better understand the impact of the electrostatic effect of the to G⁻ on the tunability of the electronic character of internucleotidic phosphates in the heptameric ssRNAs 5b, 6b, 7b and 8b, we have also performed their alkaline hydrolysis at pH 12.5 at 20 °C, and have identified the preferences of the cleavage sites at various phosphates, which are p₂, p₃ and p₄ (Fig. 3). The results of these alkaline hydrolysis studies have been compared with the hydrolysis of analogous N^1-Me- heptameric ssRNA sequences 5c, 7c and 8c under identical conditions in order to establish the role of the electrostatic effect of the 9-guanylate ion (and the 2′-OH to 2-O⁻ ionization) on the internucleotidic phosphate. It turned out that the relative alkaline hydrolysis rate at those particular phosphates (p₂, p₃ and p₄) in the N^1-Me- heptamers was reduced from 16–78% compared to those in the native counterparts [Fig. 4, and ESI 2 (Fig. S11)]. Thus, these physico-chemical studies have shown that those p₂, p₃ and p₄ phosphates in the native heptameric RNAs, which show pK_a2 as well as more deshielding (owing to weaker ³¹P screening) in the alkaline pH compared to those at the neutral pH, are more prone to the alkaline hydrolysis because of their relatively enhanced electrophilic character resulting from weaker ³¹P screening. This screening effect originates as a result of the systematic charge repulsion effect between the electron cloud in the outermost orbitals of phosphorus and the central guanylate ion, leading to delocalization of the phosphorus pπ charge into its dπ orbitals. It is thus likely that, just as in the non-enzymatic hydrolysis, the enzymatic hydrolysis of a specific phosphate in RNA by general base-catalysis in RNA-cleaving proteins (RNase A, RNA phosphodiesterase or nuclease) can potentially be electrostatically influenced by tuning the transient charge on the nucleobase in the steric proximity or as a result of specific sequence context owing to nearest-neighbor interactions.