Cleavage of Highly Structured Viral RNA Molecules by Combinatorial Libraries of Hairpin Ribozymes

Abstract

Combinatorial libraries of hairpin ribozymes representing all possible cleavage specificities (>10 5 ) were used to evaluate all ribozyme cleavage sites within a large (4.2-kilobase) and highly structured viral mRNA, the 26 S subgenomic RNA of Sindbis virus. The combinatorial approach simultaneously accounts for target site structure and dynamics, together with ribozyme folding, and the sequences that result in a ribozymesubstrate complex with maximal activity. Primer extension was used to map and rank the relative activities of the ribozyme pool against individual sites and revealed two striking findings. First, only a small fraction of potential recognition sites are effectively cleaved (activityselected sites). Second, nearly all of the most effectively cleaved sites deviated substantially from the established consensus selection rules for the hairpin ribozyme and were not predicted by examining the sequence, or through the use of computer-assisted predictions of RNA secondary structure. In vitro selection methods were used to isolate ribozymes with increased activity against substrates that deviate from the GUC consensus sequence. trans-Acting ribozymes targeting nine of the activity-selected sites were synthesized, together with ribozymes targeting four sites with a perfect match to the cleavage site consensus (sequence-selected sites). Activity-selected ribozymes have much higher cleavage activity against the long, structured RNA molecules than do sequence-selected ribozymes, although the latter are effective in cleaving oligoribonucleotides, as predicted. These results imply that, for Sindbis virus 26 S RNA, designing ribozymes based on matches to the consensus sequence may be an ineffective strategy.Small, trans-acting ribozymes including the hairpin ribozyme and the hammerhead ribozyme function as sequenceselective ribonucleases. Because their sequence specificity can be manipulated in the laboratory, ribozymes hold considerable promise as tools for targeted RNA inactivation within cells and organisms. Numerous potential applications for ribozymes are being actively explored, including the analysis of gene function, and the development of therapeutics for genetic and viral diseases.The hairpin ribozyme is derived from the minus strand of the satellite RNA of tobacco ringspot virus (1, 2) and catalyzes a reversible site-specific RNA cleavage reaction in the presence of Mg 2ϩ to yield products with 5Ј-hydroxyl and 2Ј,3Ј-cyclic phosphate termini (3-4). The ribozyme folds into two domains, termed A and B, which must interact for cleavage to occur (Fig.

E6AP gene suppression and characterization with in vitro selected hammerhead ribozymes

“…Combinatorial screening methods present one of the most efficient means of examining every potential site in a minimal number of reactions. This approach has been used in defining accessible sites in vitro for antisense oligonucleotides, 29 ribozymes [13][14][15] and catalytic DNA. 20 Lieber and Strauss 18 first reported the use of hammerhead ribozymes in a nondirected approach to target site selection.…”

“…Toward this end, an empirical approach represents a more accurate means of identifying the most effective cleavage site(s) for ribozymes. Among several different approaches, [13][14][15][16][17] Lieber and Strauss 18 used a ribozyme library carrying a conserved catalytic domain and randomized hybridizing arm sequences, and 5 0 RACE to detect the most accessible sites in the human growth hormone transcript. Despite the elegant principle that…”

RNase P Ribozymes Selected in Vitro to Cleave a Viral mRNA Effectively Inhibit Its Expression in Cell Culture

“…Extensive studies have been carried out to develop strategies to express these ribozymes that colocalize with the substrates within a particular cellular compartment, and to design ribozymes to target the mRNA regions that are accessible to binding (4,9,(21)(22)(23)(24). These studies have led to significant improvements of the efficacies of hammerhead and hairpin ribozymes in cellular environments.…”

Semliki Forest virus biodistribution in tumor-free and 4T1 mammary tumor-bearing mice: a comparison of transgene delivery by recombinant virus particles and naked RNA replicon

“…In contrast, alphavirus replicon RNA probably has an increased stability because of its secondary structure, which may protect it from degradation. 38 In addition to high expression levels of inserted gene provided by self-replicating RNA, the various RNAspecies produced by the replicon amplification initiates antiviral program resulting in type I interferon production and induction of apoptosis. 6,39 Therefore, the RNA-based vectors are potent immunostimulatory ligands providing excellent adjuvant properties and inducing strong cytopathic effect.…”