Synthesis of an amplifiable reporter RNA for bioassays

Abstract

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 Qβ RHA polymerase in vitro. This RNA can be amplified exponentially, with a population doubling time of 36 seconds, resulting in the synthesis of 10⁶ copies of each molecule in 12 minutes. We have used chemioal methods to attach blotin to the 5′ terminus of midivariant RNA via a disulfide linker. This biotinylated RNA combines with avidin to give a produot that is readily purified by gel electrophoresis. The RNA-bio-tin-avidin adduct, and the RNA released from it by reductive cleavage of the linker arm, replicate normally. The RHA-biotin-avidin adduot should be a suitable reporter for a variety of replication-assisted bioassays involving biotinylated antibodies or biotinylated nucleic acid probes.