Implications of ribozyme kinetics for targeting the cleavage of specific RNA molecules in vivo: more isn't always better

Abstract

Kinetic and thermodynamic factors that determine specificity of RNA cleavage by ribozymes are illustrated with examples from recent work with a ribozyme derived from the group I intron of Tetrahymena thermophila pre-rRNA. The conclusions also apply to other ribozymes, to antisense oligonucleotide experiments, and to RNA and DNA cleavage agents that can recognize a single-stranded or double-stranded region of variable length. At first, adding bases to a ribozyme's recognition sequence is expected to increase cleavage of the target RNA relative to cleavage of other RNAs. However, adding more bases ultimately reduces this discrimination, as cleavage occurs essentially every time the target RNA or a mismatched RNA binds the ribozyme. This occurs despite the weaker binding of the mismatched RNA because dissociation becomes too slow (binding is too strong) to allow the ribozyme to "choose" between cleavage of the target RNA and a mismatched RNA. In summary, more (base pairing) isn't always better, because maximal discrimination requires equilibrium binding prior to cleavage. The maximum discrimination that can be obtained is expected to be greater with an A + U-rich recognition sequence than with a G + C-rich recognition sequence. This is because the weaker A.U base pairs (relative to G-C base pairs) allow recognition to be spread over a larger number of bases while preventing binding that is too strong. Finally, creating an A-rich ribozyme rather than a U-rich ribozyme avoids the loss in discrimination expected with U-rich ribozymes from the formation of U.G wobble pairs in addition to the "targeted" Watson-Crick U.A pair.