RESUMEN
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.
Asunto(s)
Avidina/análogos & derivados , Biotina/análogos & derivados , Ovalbúmina/análogos & derivados , ARN/análogos & derivados , Avidina/síntesis química , Secuencia de Bases , Bioensayo , Biotina/síntesis química , ARN Polimerasas Dirigidas por ADN/metabolismo , Indicadores y Reactivos , Mutación , Conformación de Ácido Nucleico , Radioisótopos de Fósforo , Fosforilación , Q beta Replicasa/metabolismo , ARN/síntesis química , Ribonucleasa T1Asunto(s)
Exodesoxirribonucleasas/metabolismo , Guanosina Trifosfato/análogos & derivados , Neoplasias Hepáticas Experimentales/análisis , ARN/farmacología , Saccharomyces cerevisiae/enzimología , Animales , Electroforesis en Gel de Poliacrilamida , Exodesoxirribonucleasa V , Guanosina Trifosfato/metabolismo , Poli A/metabolismo , Polirribonucleótidos/metabolismo , ARN/análogos & derivados , ARN/metabolismo , ARN Nuclear PequeñoRESUMEN
1. The interaction of ribonuclease U-2 (RNAase U-2) with its substrate analogues has been investigated by a gel filtration method. At pH 4.5 and 30 degrees C, the apparent binding strength of the substrate analogues was in the following order; adenylate greater than guanylate greater than inosylate greater than cytidylate among 2'-nucleotides and 2'- greater than 3'- greater than 5'- among adenylate isomers. The formation of an equimolar complex of RNAase U-2 and 2'-nucleotide was indicated from the Scatchard plot. 2. The interaction of RNAase U-2 with 2'-adenylate or 2'-guanylate was observed spectrophotometrically. The complex of RNAase U-2 and 2'-adenylate yielded not only an absorption difference spectrum having a broad positive peak at 280 to 285 nm and a negative trough at 256 nm but also a circular dichroic difference spectrum having a positive peak at around 250 nm and a negative trough at around 290 nm. The complex of RNAase U-2 and 2'-guanylate gave a similar difference spectrum to that of the RNAase T-1 - 3'-guanylate complex, in absorption as well as in circular dichroism.