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