| Autor: |
Vaish, Narendra K, Jadhav, Vasant R, Kossen, Karl, Pasko, Christopher, Andrews, Lori E, McSwiggen, James A, Polisky, Barry, Seiwert, Scott D |
| Zdroj: |
RNA; September 2003, Vol. 9 Issue: 9 p1058-1072, 15p |
| Abstrakt: |
We describe a strategy for the ultra-sensitive detection of nucleic acids using "half" ribozymes that are devoid of catalytic activity unless completed by a trans-acting target nucleic acid. The half-ribozyme concept was initially demonstrated using a construct derived from a multiple turnover Class I ligase. Iterative RNA selection was carried out to evolve this half-ribozyme into one activated by a conserved sequence present in the hepatitis C virus (HCV) genome. Following sequence optimization of substrate RNAs, this HCV-activated half-ribozyme displayed a maximal turnover rate of 69 min(-1) (pH 8.3) and was induced in rate by approximately 2.6 x 10(9)-fold by the HCV target. It detected the HCV target oligonucleotide in the zeptomole range (6700 molecules), a sensitivity of detection roughly 2.6 x 10(6)-fold greater than that previously demonstrated by oligonucleotide-activated ribozymes, and one that is sufficient for molecular diagnostic applications. |
| Databáze: |
Supplemental Index |
| Externí odkaz: |
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