Improved Approach To Rna And Protein Recognition For Pathogen Detection

We are developing (U.S. Air Force, SBIR) a proprietary technology, based on the enzyme Q-beta replicase, to very rapidly detect and identify microorganisms (rRNA), virions (particularly RNA-based), and proteins of interest for pathogen detection. This enzyme is known to amplify a specific RNA signal one billion-fold in less than fifteen minutes at a constant temperature of 37 degreesC. RNA probes are made in "halves" that are joined to each other after their terminal ends are brought into proximity mediated by the binding to the target. Only such a full-length molecule can be amplified. We have demonstrated specific examples of recognition of RNA target sequences of interest in species of Bacillus and are investigating methods for overcoming the well-known promiscuity of the enzyme so that this recognition feature can be utilized with confidence, even in the presence of dirty backgrounds.In applying a variation of this technology to protein recognition, we have demonstrated specific steps in the process of recognition of the viral, HIV-1 Rev protein. To this end, a well-characterized RNA sequence that is specifically bound by Rev protein, known as the Rev-responsive element (RRE), was cloned into our new RNA template. This composite RNA binds Rev protein specifically and maintains an efficient rate of amplification. Two RNA detector molecules were made from this RNA by effectively separating the molecule in half by PCR and transcribing RNAs that each contain one half of the RRE and one half of the replicable vector RNA. We have recently demonstrated that these "halves" can specifically and tightly bind to Rev protein. We are currently optimizing the efficacy of detection of this complex.