Inhibition of Rev·RRE complexation by triplex tethered oligonucleotide probes

We have described a class of molecules, called tethered oligonucleotide probes (TOPs), that bind RNA on the basis of both sequence and structure. TOPs consist of two short oligonucleotides joined by a tether whose length and composition may be varied using chemical synthesis. In a triplex TOP, one oligonucleotide recognizes a short single-stranded region in a target RNA through the formation of Watson-Crick base pairs; the other oligonucleotide recognizes a short double-stranded region through the formation of Hoogsteen base pairs. Binding of triplex TOPs to an HIV-1 Rev Response Element RNA variant (RRE(AU)) was measured by competition electrophoretic mobility shift analysis. Tripler TOP·RRE(AU) stabilities ranged between -9.6 and -6.1 kcal mol^-1 under physiological conditions of pH, salt, and temperature. Although the most stable triplex TOP·RRE(AU) complex contained 12 contiguous U·AU triple helical base pairs, complexes containing only six or nine triple helical base pairs also formed. Tripler TOPs inhibited formation of the RRE·ReV complex with IC₅₀ values that paralleled the dissociation constants of the analogous triplex TOP·RRE(AU) complexes. In contrast to results obtained with TOPs that target two single-stranded RRE regions, inhibition of ReV·RRE(AU) complexation by triplex TOPs did not require pre-incubation of RRE(AU) and a TOP: triplex TOPs competed efficiently with Rev for RRE(AU) and inhibited RREAU·ReV complexation at equilibrium.