Amplifiable hybridization probes.

Abstract

Amplifiable hybridization probes enable the development of extremely sensitive clinical assays. These novel molecules consist of a probe sequence embedded within the sequences of a replicatable RNA. The molecules are first hybridized to target sequences in a conventional manner. The probe-target complexes are then isolated and the probes are released from their targets. The released probes are then amplified by incubation with the RNA-directed RNA polymerase, Q-beta replicase. The replicase copies the probes in a geometrically increasing manner: after each round of copying, the number of RNA molecules is twice the previous number. The doubling process is very rapid, resulting in as many as one billion copies of each molecule in 30 minutes. The amount of RNA that is made is large enough to be measured without using radioisotopes. Theoretically, these assays should be extraordinarily sensitive, since only one probe molecule is required to start the amplification process. In practice the sensitivity of the assays is limited by the presence of non hybridized probes that persist, despite extensive washing of the probe-target hybrids. Currently, the limit of detection is about 10,000 molecules of target. However, replicatable probes are now being prepared that include a "molecular switch", which is a region of the RNA that undergoes a conformational change when the probe sequence hybridizes to its target. Protocols are being developed that link signal generation to the state of this switch. The simplicity and speed of the enzymatic steps that are required facilitate automation of the assays.