Kaukinen, Ulla:
The Effect of Base Sequence and Secondary Structure on the Reactivity of RNA Phosphodiester Bonds
Doctoral thesis, University of Turku, Finland 2004
Annales Universitatis Turkuensis AI 314
Abstract:
RNA phosphodiester bonds are cleaved by an intramolecular transesterification reaction initiated by an attack of the neighbouring hydroxy group on the phosphorus atom. This leads to the formation of a pentacoordinated phosphorane species. The subsequent departure of the 5'-linked nucleoside results in RNA strand scission and the formation of RNA fragments with a 3'-terminal 2',3'-cyclic phosphate group and a free 5'-terminal hydroxy group. Detailed knowledge of the factors that govern the reactivity of phosphodiester bonds is particularly important for understanding the catalytic efficiency of small ribozymes, as well as for the development of artificial nucleases. Although the reaction mechanism is rather well known, little is known of the effects of base sequence and secondary structure on the rate of phosphodiester bond cleavage. The aim of this thesis was to gain insight into the various structural factors that affect the reactivity. Toward this goal, the reactivity of phosphodiester bonds within linear single-stranded oligonucleotides of varying base sequence and within different secondary structures was examined experimentally, and the structures of these compounds were investigated theoretically.
The results indicate that in linear single-stranded oligonucleotides, the reactivity of one particular phosphodiester bond is strongly dependent on the base sequence of the entire oligomer. Both rate retardation and acceleration were observed, relative to the reactivity of a fully flexible phosphodiester bond. With respect to secondary structures, phosphodiester bonds close to double-helical regions were more stable than those in the middle of single-stranded regions of the secondary structure. Comparison of the structural data obtained from theoretical calculations with experimentally determined rate constants indicates that the hydrolytic stability of phosphodiester bonds is related to the rigidity of the sugar-phosphate backbone near the cleavage site. In single-stranded oligonucleotides, the backbone stabilisation is due mainly to the vertical interactions between bases (base-stacking), whereas in secondary structures, both double-helixes and base-stacking stabilise the backbone. When the backbone is more flexible, the reactivity might approach that of the fully flexible reference compound. If the transition state (TS) is somehow stabilised, e.g., due to enhanced base-stacking, rate acceleration might occur. In addition, solvation of the cleavage site both in the initial state and TS affects the reactivity. In metal ion-promoted cleavage, the good coordination of metal ions to the cleavage site appears to compensate for the loss of inherent reactivity that results from the increased rigidity of the backbone. In summary, the cleavage of phosphodiester bonds within polymeric RNA molecules is a complicated phenomenon that is affected by several factors.