Enhancing the Second Step of the Trans Excision-Splicing Reaction of a Group I Ribozyme by Exploiting P9.0 and P10 for Intermolecular Recognition

Abstract

We previously reported that a group I intron-derived ribozyme can catalyze the excision of targeted sequences from within RNAs in vitro and that dissociation of the bridge-3' exon intermediate between the two reaction steps is a significant contributing factor to low product yields. We now analyze the effects of increasing the length, and thus the strength, of helices P9.0 and P10, which occur between the ribozyme and the bridge-3' exon region of the substrate, on this trans excision-splicing reaction. Using substrates where lengthy targeted regions are excised, these modifications can significantly increase product yields, specifically by enhancing the second reaction step. A threshold for product formation is obtained, however, at around five base pairs for P10 and eight base pairs for P9.0. Nevertheless, elongating P9.0 appears to be the more effective strategy, as both substrate binding and the rate of the second reaction step increase. In addition, P10 is required when P9.0 is not elongated. Also, a strong P9.0 helix cannot replace a weaker P10 helix, indicating that P9.0 and P10 play somewhat distinct roles in the reaction. We also show that second-step inhibition stems from the formation of an extended P1 helix (P1ex), consisting of as little as a single Watson-Crick base pair, as well as the mere presence of substrate nucleosides immediately downstream from P10. Both of these inhibitory components can be overcome by utilizing P9.0 and P10 elongated ribozymes. This work sets forth an initial framework for rationally designing more effective trans excision-splicing ribozymes.

Computational prediction of efficient splice sites for trans-splicing ribozymes

“…3B) may be the faster transsplicing kinetics with short substrates. Several processes may play a stronger role at faster reaction kinetics, such as the initial folding of the ribozyme (Pan et al 1997;Shi et al 2009) and later steps in the trans-splicing process (Karbstein et al 2002;Bell et al 2004), which are not captured by the thermodynamic model used in this study.…”

In vivo excision of a single targeted nucleotide from an mRNA by a trans excision-splicing ribozyme

“…The design of the in vivo TES test system was based on work involving the trans-splicing ribozyme (Sullenger and Cech 1994;Jones et al 1996;Jones and Sullenger 1997;Phylactou et al 1998;Kohler et al 1999;Rogers et al 2002;Byun et al 2003;Ryu et al 2003;Kastanos et al 2004;Shin et al 2004), as well as design principles garnered through studies of the TES ribozyme in vitro (Bell et al 2002(Bell et al , 2004. Our simplest TES reaction system involves the removal of a single nucleotide from a substrate, so we designed a test system in which we create a single base insertion mutation in the coding region of GFP ( Fig.…”

“…In all the test systems, the length of recognition element 1 (RE1) was maintained at six bases. Previous work on the TES ribozyme indicated that increasing the length of RE3 can be beneficial to the reaction (Bell et al 2004), while work with the trans-splicing ribozyme showed a requirement for increased 3 0 exon interactions for in vivo activity (Kohler et al 1999;Rogers et al 2002;Byun et al 2003;Ryu et al 2003;Kastanos et al 2004;Shin et al 2004). Thus, ribozymes were tested with RE3 lengths of three bases (native length), five bases, and seven bases.…”

“…Targeted ribozymes (GFP rP3X) were tested with RE3 lengths of three, five, and seven bases. Increasing the length of RE3 increases the interaction between the ribozyme and the 3 0 exon of the substrate, which helps prevent 3 0 exon dissociation prior to the second reaction step and can improve TES product formation in vitro (Bell et al 2004). The native form of the ribozyme (rP3X) served as a non-GFP-targeted control for ribozyme activity, while inactive forms of the ribozymes (containing a four-base deletion in the guanosine binding site) were used as negative controls.…”

“…1). In vitro, this ribozyme has been used to excise inserts from 1 to 28 nucleotides in length and can be re-engineered to target nonnative sequences, including the flanking region of the trinucleotide repeat that causes myotonic dystrophy (Bell et al 2002(Bell et al , 2004. The usefulness of these ribozymes, however, will be greatly enhanced by demonstrating in vivo reactivity.…”