title: Zeptomole detection of a viral nucleic acid using a target-activated ribozyme.
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.
authors.affiliation: Sirna Therapeutics, Inc., Boulder, Colorado 80301, USA. vaishn@sirna.com
authors.forename: Narendra K
authors.initials: NK
authors.lastname: Vaish
authors.forename: Vasant R
authors.initials: VR
authors.lastname: Jadhav
authors.forename: Karl
authors.initials: K
authors.lastname: Kossen
authors.forename: Christopher
authors.initials: C
authors.lastname: Pasko
authors.forename: Lori E
authors.initials: LE
authors.lastname: Andrews
authors.forename: James A
authors.initials: JA
authors.lastname: McSwiggen
authors.forename: Barry
authors.initials: B
authors.lastname: Polisky
authors.forename: Scott D
authors.initials: SD
authors.lastname: Seiwert
mesh_terms: D016174:Hepacivirus / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000032:analysis*; D013997:Time Factors
chemical_list: D016337:RNA, Catalytic; D012367:RNA, Viral