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 (RREAU) was measured by competition electrophoretic mobility shift analysis. Triplex TOP.RREAU 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.RREAU complex contained 12 contiguous U.AU triple helical base pairs, complexes containing only six or nine triple helical base pairs also formed. Triplex TOPs inhibited formation of the RRE.Rev complex with IC50 values that paralleled the dissociation constants of the analogous triplex TOP.RREAU complexes. In contrast to results obtained with TOPs that target two single-stranded RRE regions, inhibition of Rev.RREAU complexation by triplex TOPs did not require pre-incubation of RREAU and a TOP: triplex TOPs competed efficiently with Rev for RREAU and inhibited RREAU.Rev complexation at equilibrium.
Moses
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AC
Department of Chemistry, Yale University, New Haven, CT 06520-8107, USA.
Huang
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SW
Schepartz
A
A
D001482:Base Composition; D001483:Base Sequence; D015694:Gene Products, rev / Q000187:drug effects* / Q000378:metabolism*; D015752:Genes, env; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D012313:RNA / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D012367:RNA, Viral / Q000187:drug effects / Q000378:metabolism; D013696:Temperature
D015694:Gene Products, rev; D015345:Oligonucleotide Probes; D012367:RNA, Viral; D012313:RNA