Extremely sensitive assays will be developed for the detection of pathogenic human retroviruses. The assays will be designed for the screening of donated blood and for the identification of asymptomatic carriers, in order to prevent the spread of T-lymphotropic leukemia/lymphoma and acquired immunodeficiency syndrome (AIDS). Novel recombinant RNA molecules will be prepared. They will serve as specific hybridization probes for retroviral sequences and will also serve as exponentially amplifiable reporters. Incubation of the hybrids with Q beta replicase will generate as many as one billion RNA copies of each probe, enabling the measurement of the number of retroviral targets in the sample. The assays will not require the fractionation of cells or cellular components; hybridization will take place in solution; hybrids will be separated from unhybridized probes on magnetic beads; amplification will require only a single incubation at 37C; and the entire assay will take less than three hours. Assay formats will be developed that will enable the simultaneous detection of different pathogenic retroviruses in the same sample. Although the use of exponentially amplifiable probes theoretically allows the detection of as little as one target molecule, the actual sensitivity of these assays will be determined by the number of non-specifically bound probes that persist, despite extensive washing of the hybrids. In order to eliminate, or markedly reduce, this source of background, the probe molecules will be altered to contain a molecular switch. This is a region of the molecule that undergoes a conformational change when the probe hybridizes to its target. Non-specifically bound probes will not undergo this change. An additional enzymatic step will then be added to the assay that will obligately link the replication of RNA reporters to the state of the molecular switch. The use of binary probes in assays will also be explored. Two different single-stranded DNA probes will be prepared. Each will comprise a different portion of a template for the transcription of replicatable RNA reporters. The probes will not be able to function as a template by themselves: they will first have to hybridize to adjacent regions of the target sequence, where they will be joined together by incubation with DNA ligase. Since DNA ligase only works with hybridized DNAs, the generation of replicatable RNA reporters from these templates will be dependent on the hybridization of the probes to the target.
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