Testing a cell-based selex approach to identify hammerhead ribozymes that suppress human rhodopsin expression
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SELEX (Systematic Evolution of Ligands by Exponential Enrichment) is an in vitro method to identify small agents (eg. oligonucleotides, peptides, RNAs) that can interact with and bind to a larger molecular target (eg. protein, mRNA). Cell SELEX was designed as a scientific approach to utilize cell-based evolutionary principles to identify post-transcriptional gene silencing ( PTGS ) agents that are effective in the intracellular milieu. The intent of Cell SELEX was to identify PTGS agents (eg. hammerhead ribozymes) that function to suppress expression of a target mRNA and protein within the complexity of human cells. In Cell SELEX, evolutionary principles of mutation (combinatorial PTGS expression library generation), selection (target mRNA or protein construct is toxic, so cells die unless they have PTGS agents that can reduce target expression), and amplification of PTGS expression plasmids (amplification of surviving mammalian cells and subsequent amplification of plasmids in bacteria) are used. To conduct a limited proof-of-principle test for Cell SELEX, we first designed and constructed a rhodopsin-specific GUC-specific hammerhead ribozyme sub-library. The target gene (human rhodopsin) was made toxic by an in-frame fusion to thymidine kinase ( TK ) cDNA. The rhodopsin-TK fusion protein expressed from a Rhodopsin-TK fusion mRNA is toxic in cells when a prodrug, ganciclovir ( GCV ), is added. Toxic target gene expression followed by GCV addition leads to death of human cells. This permits a strong selection approach to screen for PTGS agents able to suppress expression of the target gene at the post-transcriptonal level. The ribozyme expression library was screened in HEK293E mammalian cells and a subset of hammerhead ribozyme expression constructs was identified against our target rhodopsin mRNA. Specifically, we obtained hammerhead ribozymes from the sub-library that rescued cells from death due to the engineered toxic target gene. Successful ribozyme expression plasmids were amplified in surviving cells and then in bacteria and their identity determined by DNA sequencing. These results suggest that Cell SELEX may be a powerful tool for the isolation of active hammerhead ribozymes against specific targets, which might not otherwise be identified using standard rational PTGS design principles.