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Use of the Fluorescent Nucleoside Analogue Benzo[g]quinazoline 2'-O-Methyl-b-D-ribofuranoside to Monitor the Binding of the HIV-1 Tat Protein or of Antisense Oligonucleotides to the TAR RNA Stem-Loop
The Tat protein is an essential trans-activator of HIV gene expression. It interacts with its RNA recognition sequence, the trans-activation responsive region TAR, as well as cellular factors. These interactions are potential targets for drug discovery against HIV infection. We have developed a new and sensitive assay for the measurement of Tat binding to TAR in solution under equilibrium conditions based on the change of fluorescence of the base analogue benzo[g]quinazoline-2,4(1H,3H)-dione (BgQ) incorporated into the chemically synthesized model TAR stem-loop 2 to which was added Tat-[37-72] peptide (3). The results show that Tat-TAR binding strength is 2 - 3-fold stronger than has previously been determined by mobility-shift analysis. Changes of fluorescence were used also to measure the binding of antisense 2-O-methyloligonucleotides to TAR 2.