Chu B C, Kramer F R, Orgel L E
Nucleic Acids Res. 1986 Jul 25;14(14):5591-603. doi: 10.1093/nar/14.14.5591.
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.
用可扩增的报告分子取代诸如荧光分子或酶等报告基团，应能使生物测定的灵敏度大大提高，因为在短时间内可以积累大量的报告分子拷贝。中变体RNA是一种合适的报告分子，因为它在体外可被QβRNA聚合酶自催化复制。这种RNA可以呈指数级扩增，群体倍增时间为36秒，从而在12分钟内合成每个分子的10⁶个拷贝。我们已使用化学方法通过二硫键接头将生物素连接到中变体RNA的5'末端。这种生物素化的RNA与抗生物素蛋白结合，得到的产物可通过凝胶电泳轻松纯化。RNA-生物素-抗生物素蛋白加合物，以及通过接头臂的还原裂解从中释放的RNA，能正常复制。RNA-生物素-抗生物素蛋白加合物应该是适用于各种涉及生物素化抗体或生物素化核酸探针的复制辅助生物测定的报告分子。
