Synthesis of an amplifiable reporter RNA for bioassays.
The replacement of reporter groups, such as fluorescent molecules or enzymes, by an amplifiable reporter should lead to bioassays of greatly increased sensitivity, since a very large number of copies of the reporter can be accumulated in a short time. Midivariant RNA is an appropriate reporter, since it is autocatalytically replicated by Q beta RNA polymerase in vitro. This RNA can be amplified exponentially, with a population doubling time of 36 seconds, resulting in the synthesis of 10(6) copies of each molecule in 12 minutes. We have used chemical methods to attach biotin to the 5' terminus of midivariant RNA via a disulfide linker. This biotinylated RNA combines with avidin to give a product that is readily purified by gel electrophoresis. The RNA-biotin-avidin adduct, and the RNA released from it by reductive cleavage of the linker arm, replicate normally. The RNA-biotin-avidin adduct should be a suitable reporter for a variety of replication-assisted bioassays involving biotinylated antibodies or biotinylated nucleic acid probes.
D001360:Avidin / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D001483:Base Sequence; D001681:Biological Assay; D001710:Biotin / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D007202:Indicators and Reagents; D009154:Mutation; D009690:Nucleic Acid Conformation; D010047:Ovalbumin / Q000031:analogs & derivatives*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011777:Q beta Replicase / Q000378:metabolism; D012313:RNA / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D006163:Ribonuclease T1
D007202:Indicators and Reagents; D010761:Phosphorus Radioisotopes; C049779:RNA-biotin-avidin-adduct; D001360:Avidin; D012313:RNA; D001710:Biotin; D010047:Ovalbumin; D011777:Q beta Replicase; D012321:DNA-Directed RNA Polymerases; D006163:Ribonuclease T1