Use of the fluorescent nucleoside analogue benzo[g]quinazoline 2 '-O-methyl-beta-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 responsiv e 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 u nder 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 st ronger than has previously been determined by mobility-shift analysis. Chan ges of fluorescence were used also to measure the binding of antisense 2'-O -methyloligonucleotides to TAR 2.