R Yamamoto1, T Baba, P K Kumar. 1. National Institute of Bioscience and Human Technology, University of Tsukuba, Tsukuba 305-8572, Japan.
Abstract
BACKGROUND: We reported an aptamer, RNATat that binds to the Tat protein of HIV with two orders of magnitude greater (133-fold) affinity over the TAR RNA of HIV-1 and specifically inhibits the Tat-dependent trans-activation of transcription, both in vitro and in vivo (demonstrated in the accompanying article, Yamamoto et al., this issue pp. 371-388). We now report the use of aptamer-derived oligomers to analyze the Tat of HIV and the possible applications of such constructs in the field of biosensors. RESULTS: To make new molecular beacon, we constructed two RNA oligomers that derived from RNATat. To one of the split RNA oligomers that forms a hairpin structure, the fluorophore and quencher were attached at the 5'- and 3'-ends, respectively. Specifically in the presence of Tat or its peptides, but not in the presence of other RNA binding proteins, the two oligomers undergo a conformational change to form a duplex that leads to relieving of fluorophore from the quencher, and thus a significant enhancement of the fluorescence of fluorescein was observed. CONCLUSION: A novel strategy for exploiting aptamers in the analysis of Tat (analyte) has been described. A similar strategy could be used to study other analytes such as proteins and small molecules. In addition, the molecular beacon aptamer requires half the length of target sequence (eight nucleotides) in comparison with molecular beacons. Thus, it is conceivable that we could insert an analyte-binding site into molecular beacons to convert them to signalling beacons.
BACKGROUND: We reported an aptamer, RNATat that binds to the Tat protein of HIV with two orders of magnitude greater (133-fold) affinity over the TAR RNA of HIV-1 and specifically inhibits the Tat-dependent trans-activation of transcription, both in vitro and in vivo (demonstrated in the accompanying article, Yamamoto et al., this issue pp. 371-388). We now report the use of aptamer-derived oligomers to analyze the Tat of HIV and the possible applications of such constructs in the field of biosensors. RESULTS: To make new molecular beacon, we constructed two RNA oligomers that derived from RNATat. To one of the split RNA oligomers that forms a hairpin structure, the fluorophore and quencher were attached at the 5'- and 3'-ends, respectively. Specifically in the presence of Tat or its peptides, but not in the presence of other RNA binding proteins, the two oligomers undergo a conformational change to form a duplex that leads to relieving of fluorophore from the quencher, and thus a significant enhancement of the fluorescence of fluorescein was observed. CONCLUSION: A novel strategy for exploiting aptamers in the analysis of Tat (analyte) has been described. A similar strategy could be used to study other analytes such as proteins and small molecules. In addition, the molecular beacon aptamer requires half the length of target sequence (eight nucleotides) in comparison with molecular beacons. Thus, it is conceivable that we could insert an analyte-binding site into molecular beacons to convert them to signalling beacons.
Authors: Chaoyong James Yang; Steffen Jockusch; Marie Vicens; Nicholas J Turro; Weihong Tan Journal: Proc Natl Acad Sci U S A Date: 2005-11-21 Impact factor: 11.205