Computational prediction of efficient splice sites for trans-splicing ribozymes

TitleComputational prediction of efficient splice sites for trans-splicing ribozymes
Publication TypeJournal Article
Year of Publication2012
AuthorsMeluzzi, D, Olson, KE, Dolan, GF, Arya, G, and Muller, UF
JournalRNA (New York, N.Y.)
Volume18
Issue3
Start Page590
Pagination590 - 602
Date Published03/2012
Abstract

Group I introns have been engineered into trans-splicing ribozymes capable of replacing the 3'-terminal portion of an external mRNA with their own 3'-exon. Although this design makes trans-splicing ribozymes potentially useful for therapeutic application, their trans-splicing efficiency is usually too low for medical use. One factor that strongly influences trans-splicing efficiency is the position of the target splice site on the mRNA substrate. Viable splice sites are currently determined using a biochemical trans-tagging assay. Here, we propose a rapid and inexpensive alternative approach to identify efficient splice sites. This approach involves the computation of the binding free energies between ribozyme and mRNA substrate. We found that the computed binding free energies correlate well with the trans-splicing efficiency experimentally determined at 18 different splice sites on the mRNA of chloramphenicol acetyl transferase. In contrast, our results from the trans-tagging assay correlate less well with measured trans-splicing efficiency. The computed free energy components suggest that splice site efficiency depends on the following secondary structure rearrangements: hybridization of the ribozyme's internal guide sequence (IGS) with mRNA substrate (most important), unfolding of substrate proximal to the splice site, and release of the IGS from the 3'-exon (least important). The proposed computational approach can also be extended to fulfill additional design requirements of efficient trans-splicing ribozymes, such as the optimization of 3'-exon and extended guide sequences.

DOI10.1261/rna.029884.111
Short TitleRNA (New York, N.Y.)