High-throughput ribozyme-based assays for detection of viral nucleic acids.
Many reports have suggested that target-activated ribozymes hold potential value as detection reagents. We show that a "half"-ribozyme ligase is activated similarly by three unstructured oligoribonucleotides representing the major sequence variants of a hepatitis C virus 5'-untranslated region (5'-UTR) target and by a structured RNA corresponding to the entire 5'-UTR. Half-ribozyme ligation product was detected both in an ELISA-like assay and in an optical immunoassay through the use of hapten-carrying substrate RNAs. Both assay formats afford a limit of detection of approximately 1 x 10(6) HCV molecules (1.6 attomol, 330 fM), a sensitivity which compares favorably to that provided by standard immunoassays. These data suggest that target-activated ribozyme systems are a viable approach for the sensitive detection of viral nucleic acids using high-throughput platforms.
Kossen
Karl
K
Sirna Therapeutics Inc., 2950 Wilderness Place, Boulder, CO 80301 USA.
Vaish
Narendra K
NK
Jadhav
Vasant R
VR
Pasko
Christopher
C
Wang
Hong
H
Jenison
Robert
R
McSwiggen
James A
JA
Polisky
Barry
B
Seiwert
Scott D
SD
D001483:Base Sequence; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006526:Hepatitis C / Q000175:diagnosis* / Q000821:virology; D007118:Immunoassay / Q000379:methods; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000378:metabolism*; D012367:RNA, Viral / Q000032:analysis*; D012680:Sensitivity and Specificity
D016337:RNA, Catalytic; D012367:RNA, Viral