The applicant proposes to develop a clinical analytical method for HIV-1 Rev protein. The technology is based on the combination of two relatively new technologies. First is the SELEX technology which has so far identified strong binding nucleotide sequences to Rev protein. The second is the specific amplification of Q- beta replicase of RNA containing recognized sequences by this enzyme. The idea is to make two RNA apttamers that will bind to Rev in such a way that the ends are juxtapositioned for the ligation by T4 RNA ligase. Once this is done, the ligated RNA can be amplified specifically by Q-beta replicase. The amplified product would be used as quantitation of Rev protein. For the development of such a system, the applicant proposes to construct two recombinant RNA aptamers for Rev protein binding and for the template of Q-beta replicase. Then to find conditions the aptamers anneal maximally to Rev before they anneal to themselves. This will be followed by testing in ligation and amplification. If this is successful, the methods in test-kits will be tested using clinical samples.
