| Autor: |
Hampel, Arnold, Nesbitt, Steven, Tritz, Richard, Altschuler, Mitchell |
| Zdroj: |
Methods: A Companion to Methods in Enzymology; April 1993, Vol. 5 Issue: 1 p37-42, 6p |
| Abstrakt: |
The hairpin ribozyme is a catalytic RNA capable of specifically cleaving a variety of substrate RNA sequences in either a cis or a trans reaction. The trans reaction has favorable catalytic and physical properties that make this ribozyme a potentially powerful in vivo regulator of gene expression. The ribozyme itself can be engineered to specifically cleave long RNA substrates by recognizing and cleaving a 14- to 20-nt target sequence. For certain sequences the reaction is efficient under mild, near physiological, conditions; however, this efficiency varies with the substrate and must be determined for each particular target. Substrate is cleaved at the * in the N*GUC sequence. Substrates containing a SN*GUC sequence, where the S is G or C, are cleaved most efficiently. To cleave heterologous RNA the hairpin ribozyme is engineered to base pair to the sequences flanking the N*GUC. The 5' flanking sequence is fixed in length to 4 bp and the 3' flanking sequence is variable, but the best catalytic efficiency is normally obtained when it is between 6 and 10 nt long. The ribozyme efficiently cleaves this substrate to generate the corresponding 5' cleavage fragment and 3' cleavage fragment. The 5' cleavage fragment has a 2',3'-cyclic phosphate 3' terminus at the site of cleavage, and the 3' cleavage fragment has a 5'-OH at the site of cleavage. The hairpin ribozyme has been engineered and shown to cleave targets in vivo. It has successfully been used to specifically down-regulate gene expression in mammalian cells acting as a catalytic antisense moiety.Copyright 1993, 1999 Academic Press |
| Databáze: |
Supplemental Index |
| Externí odkaz: |
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