C-SPACE (cleavage-specific amplification of cDNA ends): a novel method of ribozyme-mediated gene identification

摘要

A hairpin ribozyme, RzCR2A, directed against position 323 of the hepatitis C virus 5′-untranslated region (HCV 5′-UTR) was used to establish and validate a novel method for the detection of cellular target molecules for hairpin ribozymes, termed C-SPACE (cleavage-specific amplification of cDNA ends). For C-SPACE, HeLa mRNA containing the transcript of interest was subjected to in vitro cleavage by RzCR2A in parallel with a control ribozyme, followed by reverse transcription using a modified SMART cDNA amplification method and cleavage-specific PCR analysis. C-SPACE allowed identification of the RzCR2A target transcript from a mixture containing the entire cellular mRNA while only requiring knowledge of the ribozyme binding sequence for amplification. In a similar approach, C-SPACE was used successfully to identify human 20S proteasome α-subunit PSMA7 mRNA as the cellular target RNA of Rz3′X, a ribozyme originally designed to cleave the negative strand HCV 3′-UTR. Rz3′X was found to substantially inhibit HCV internal ribosome entry site (IRES) activity and PSMA7 was subsequently confirmed to be involved in HCV IRES-mediated translation. Thereby, C-SPACE was validated as a powerful tool to rapidly identify unknown target RNAs recognized and cleaved by hairpin ribozymes.