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Nucleic Acids Research, 2001, Vol. 29, No. 19 e94
© 2001 Oxford University Press

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

Martin Krüger1, Carmela Beger1, Peter J. Welch2, Jack R. Barber2 and Flossie Wong-Staal1,2,*

1University of California San Diego School of Medicine, 0665, 9500 Gilman Drive, La Jolla, CA 92093-0665, USA and 2Immusol Inc., 10790 Roselle Street, San Diego, CA 92121, USA

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 {alpha}-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.

* To whom correspondence should be addressed at: Departments of Medicine and Biology, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0665, USA. Tel: +1 858 534 7957; Fax: +1 858 534 7743; Email: fwongstaal{at}ucsd.edu Present addresses:Martin Krüger, Department of Gastroenterology and Hepatology, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, D-30625 Hannover, GermanyCarmela Beger, Department of Paediatrics, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors


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 Mol. Cell. Biol.Home page
M. Kruger, C. Beger, P. J. Welch, J. R. Barber, M. P. Manns, and F. Wong-Staal
Involvement of Proteasome alpha -Subunit PSMA7 in Hepatitis C Virus Internal Ribosome Entry Site-Mediated Translation
Mol. Cell. Biol., December 15, 2001; 21(24): 8357 - 8364.
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