Selection of Efficient Cleavage Sites in Target RNAs by Using a Ribozyme Expression Library

Abstract

Inactivation of gene expression by antisense mechanisms in general and by ribozymes in particular is a powerful technique for studying the function of a gene product. We have designed a strategy for expression of ribozymes, for selection of accessible cleavage sites in target RNAs, and for isolation of ribozymes from a library of random sequences flanking the unique sequence of a hammerhead. The expression cassette for ribozyme genes is based on adenovirus-associated RNA. Alternatively, we used polymerase III or the T7 phage transcription machinery. The ribozyme sequences are positioned in the center of a stable stem-loop structure, allowing for a correctly folded ribozyme region within the expressed RNA. A library of ribozyme genes with random sequences of 13 nucleotides on both sides of the hammerhead was generated. As an example, ribozymes which are specific for seven sites within the mRNA or nuclear RNA of human growth hormone were selected and identified. Sequencing of ribozyme genes reamplified from the library confirmed not only the predicted cleavage sites but also the presence of different ribozyme variants in the library. In a test of the ribozyme variants for repression of growth hormone synthesis in a cellular assay, the strongest effect (more than 99% inhibition) was found for the variant with the shortest stretch of complementarity (7 and 8 nucleotides on either side) to the target RNA. This basic strategy seems to be applicable to the selection of suitable target sites and to the isolation of corresponding ribozymes for any mRNA of interest.

Intermolecular Cleavage by the Newt Ribozyme

“…Additionally, cell extract which had been treated with proteinase K was used in control experiments. These cleavage reactions in the presence of cell extract were performed in the presence of 70 mM MgCI, to inhibit ribonucleases [15]. The percentage of cleavage was not altered by the presence of the cell extract and the results were very similar whether the proteins in the extract had been hydrolyzed or not.…”

A selection system for identifying accessible sites in target RNAs

“…Experimental approaches for identification of cleavable sites offer clear advantages+ Lieber and Strauss (1995) constructed a library of hammerhead ribozymes+ These library ribozymes were targeted to a preselected triplet, and contained randomized sequences in the annealing arms, which allowed the screening of accessible sites in the target-RNA molecule+ In this case, the selected ribozymes cleaved an in vitro transcript efficiently and inhibited gene expression in cell culture; one selected ribozyme was successfully used for inhibition of growth-hormone expression in mice (Lieber & Kay, 1996)+ In a different approach, Birikh et al+ (1997b) used a completely randomized oligonucleotide (dN 10 ) in conjunction with RNase H to map sites that are accessible for oligonucleotide binding in an RNA transcript: the best ribozymes generated in this fashion was 150-fold more active than the most efficient ribozymes designed on the basis of the mFold program (Zuker & Stiegler, 1981)+ As a remarkably powerful tool, the systematic evolution of ligands by exponential enrichment (SELEX; Ellington & Szostak, 1990;Tuerk & Gold, 1990) has been used to isolate oligonucleotide sequences, socalled aptamers, with the capacity to recognize virtually any class of target molecules with high affinity and specificity, such as organic dyes, amino acids, biological cofactors, antibiotics, peptides and proteins, or even whole viruses and protozoan organisms (Pan et al+, 1995;Eaton, 1997;Osborne & Ellington, 1997;Bell et al+, 1998;Gal et al+, 1998;Kraus et al+, 1998;Yang et al+, 1998;Homann & Goringer, 1999;Wang et al+, 2000)+ Here, we have extended the SELEX method to isolate ribozyme-guide RNAs (Fig+ 2), which can aid in the location of optimal cleavage sites within any targeted RNA, based upon Watson-Crick base pairing between random guide RNAs and target RNAs under physiologic conditions (Fig+ 1B)+ Using long (partial and/or full-length) transcripts of human hepatitis B virus RNA (HBV, 917 nt long), human RNA polymerase I (the hRPA39 subunit of Pol I, 1,039 nt long) and mouse tumor suppressor gene "phosphatase and tensin homologue deleted on chromosome ten" (PTEN, 1,213 nt), we demonstrate that highly effective ribozymes can be engineered by systematically isolating a pool of ribozyme-guide RNA from a large library of random guide RNA sequences+ Ribozymes targeted to these library-selected sites efficiently cleaved long transcripts in vitro: the best showed a K cat /K m ϭ 1+0 ϫ 10 6 (M Ϫ1 min Ϫ1 ), whereas others generally showed K cat /K m values of 0+6 ϫ 10 6 (M Ϫ1 min Ϫ1 )+ Library-selected ribozymes targeted to HBV effectively inhibited viral FIGURE 1. Schematic representation of the Random Selection Library+ A: Diagram of a hammerhead ribozyme, showing a catalytic core, a random 6-nt 59-flanking region and a 9-nt 39-flanking region+ Arrowhe...…”

E6AP gene suppression and characterization with in vitro selected hammerhead ribozymes

“…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 underlies this method, its high technical demand appears to have deterred others from utilizing it.…”