Secondary structure prediction and in vitro accessibility of mRNA as tools in the selection of target sites for ribozymes

Abstract

We have investigated the relative merits of two commonly used methods for target site selection for ribozymes: secondary structure prediction (MFold program) and in vitro accessibility assays. A total of eight methylated ribozymes with DNA arms were synthesized and analyzed in a transient co-transfection assay in HeLa cells. Residual expression levels ranging from 23 to 72% were obtained with anti-PSKH1 ribozymes compared to cells transfected with an irrelevant control ribozyme. Ribozyme efficacy depended on both ribozyme concentration and the steady state expression levels of the target mRNA. Allylated ribozymes against a subset of the target sites generally displayed poorer efficacy than their methylated counterparts. This effect appeared to be influenced by in vivo accessibility of the target site. Ribozymes designed on the basis of either selection method displayed a wide range of efficacies with no significant differences in the average activities of the two groups of ribozymes. While in vitro accessibility assays had limited predictive power, there was a significant correlation between certain features of the predicted secondary structure of the target sequence and the efficacy of the corresponding ribozyme. Specifically, ribozyme efficacy appeared to be positively correlated with the presence of short stem regions and helices of low stability within their target sequences. There were no correlations with predicted free energy or loop length.

Figure 1

Schematic of the hammerhead ribozyme hybridizing to its target mRNA. The arrow indicates the position of cleavage in the mRNA. Numbering is according to the nomenclature of Hertel et al. (26). Unmodified ribonucleotides are in bold lower case, 2′-O-alkylated ribonucleotides in plain lower case, and deoxynucleotides in upper case. Phosphorothioates are indicated by asterisk, while iT denotes an inverted 3′–3′ thymidine. Bases of the GUM target triplet, where M is C or A, are boxed.

Figure 2

Excerpts of the energetically optimal secondary structure of the first 1000 bases of the pTRE-PSK-Luc transcript, as predicted by MFold version 3.0 (7,8). Target sequences of the ribozymes are highlighted. Arrows indicate position of cleavage.

Figure 3

In vitro accessibility assays. (A) Separate screening of a 1.03 kb PSKH1 in vitro transcript with triplet-specific ODN libraries. Reactions were performed with 50 nM RNA and 40 µM ODN library as detailed in Materials and Methods. Arrows indicate pairs of fragments resulting from cleavage at subsequently selected ribozyme target sites. (B) Cleavage of PSKH1 RNA (50 nM) with molar excess (200 nM) of specific ODNs against ribozyme target sites selected by oligo library screening (O-465, O-519, O-539, O-548) and structural considerations.

Figure 4

Lipofectamine-mediated co-transfection of HeLa cells with ribozyme, pTRE-PSK-Luc, and pRLuc as detailed in Materials and Methods. Eight methylated (MRz-) and three allylated (ARz-) PSKH1-specific ribozymes were analyzed together with their respective controls (MRz-TF and ARz-TF). PSKH1-dependent firefly luciferase expression was normalized to Renilla luciferase expression for each sample. Normalized expression in cells transfected with control ribozymes within each series was set at 100%. Data are averages of 4–7 independent experiments. Expression levels are indicated above the error bars (+SD) of each column.

Figure 5

Dependence of ribozyme inhibitory activity on ribozyme concentration and reporter gene expression. (A) Transfection of HeLa cells with increasing concentrations of ARz-519 or MRz-287 ribozymes. Total concentration of ribozyme is adjusted to 100 nM with control ribozyme (ARz-TF or MRz-TF) and transfections performed after the standard protocol. Data for representative experiments are shown. (B) Parallel co-transfections of HeLa cells with selected ribozymes and different reporter constructs (pTRE-PSK-Luc and pcDNA3-PSK-Luc). Relative expression levels in control cells were 20-fold higher with the pcDNA3-PSK-Luc construct compared to pTRE-PSK-Luc.