Zeptomole detection of a viral nucleic acid using a target-activated ribozyme.
RNA
; 9(9): 1058-72, 2003 Sep.
Article
de En
| MEDLINE
| ID: mdl-12923255
ABSTRACT
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.
Texte intégral:
1
Collection:
01-internacional
Base de données:
MEDLINE
Sujet principal:
ARN viral
/
ARN catalytique
/
Hepacivirus
Type d'étude:
Diagnostic_studies
Langue:
En
Journal:
RNA
Sujet du journal:
BIOLOGIA MOLECULAR
Année:
2003
Type de document:
Article
Pays d'affiliation:
États-Unis d'Amérique