Abstract
Polytailing a cDNA with terminal deoxynucleotidyltransferase (TdT) results in the addition of a homopolymeric sequence at its 3'-end. Here we describe the use of tailing in competitive RT-PCR assays to evaluate cleavage efficiency of ribozymes. Using a system that perfectly mimics intracellular cleavage, we were able to detect as few as 1% of cleaved moieties. Furthermore, employing primers overlapping the junction between tails and the cleaved RNA moiety in non-competitive assays, the sensitivity of the method could be improved to <10 fg. Using the latter protocol and reactions employing a trans -acting hairpin ribozyme targeting the nucleocapsid mRNA of the mumps virus, we were able to demonstrate ribozyme-induced cleavage.
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Selected References
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- Albuquerque-Silva J., De Vos M. J., Bollen A., Houard S. "Hairpin" and "hammerhead" ribozymes directed towards the mumps virus nucleocapsid RNA: specific cleavage of a small synthetic RNA substrate and full-length mRNA. Virus Genes. 1996;13(3):211–219. doi: 10.1007/BF00366981. [DOI] [PubMed] [Google Scholar]
- Elango N. The mumps virus nucleocapsid mRNA sequence and homology among the Paramyxoviridae proteins. Virus Res. 1989 Jan;12(1):77–86. doi: 10.1016/0168-1702(89)90055-5. [DOI] [PubMed] [Google Scholar]
- Johnston J. A., Norgren S., Annerén G., Cowburn R. F., Lannfelt L. A new quantitative solution hybridisation-RNase protection assay for APP and APLP2 mRNA. Brain Res Mol Brain Res. 1996 Dec 31;43(1-2):77–84. doi: 10.1016/s0169-328x(96)00160-x. [DOI] [PubMed] [Google Scholar]
- Jones J. T., Sullenger B. A. Evaluating and enhancing ribozyme reaction efficiency in mammalian cells. Nat Biotechnol. 1997 Sep;15(9):902–905. doi: 10.1038/nbt0997-902. [DOI] [PubMed] [Google Scholar]
- Menzo S., Bagnarelli P., Giacca M., Manzin A., Varaldo P. E., Clementi M. Absolute quantitation of viremia in human immunodeficiency virus infection by competitive reverse transcription and polymerase chain reaction. J Clin Microbiol. 1992 Jul;30(7):1752–1757. doi: 10.1128/jcm.30.7.1752-1757.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Milligan J. F., Groebe D. R., Witherell G. W., Uhlenbeck O. C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 1987 Nov 11;15(21):8783–8798. doi: 10.1093/nar/15.21.8783. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ofverstedt L. G., Hammarström K., Balgobin N., Hjertén S., Pettersson U., Chattopadhyaya J. Rapid and quantitative recovery of DNA fragments from gels by displacement electrophoresis (isotachophoresis). Biochim Biophys Acta. 1984 Jun 16;782(2):120–126. doi: 10.1016/0167-4781(84)90014-9. [DOI] [PubMed] [Google Scholar]
- Sullenger B. A., Cech T. R. Tethering ribozymes to a retroviral packaging signal for destruction of viral RNA. Science. 1993 Dec 3;262(5139):1566–1569. doi: 10.1126/science.8248806. [DOI] [PubMed] [Google Scholar]