DESIGN AND SYNTHESIS OF RNA MINIDUPLEXES VIA A SYNTHETIC LINKER APPROACH .2. GENERATION OF COVALENTLY CLOSED, DOUBLE-STRANDED CYCLIC HIV-1 TAR RNA ANALOGS WITH HIGH TAT-BINDING AFFINITY

We recently developed an approach which allows rapid generation of sho rt, double-stranded oligonucleotides whereby one end of the duplex was joined and stabilized by a synthetic linker of specific design (minid uplexes)(6). Model miniduplexes based on the HIV-1 TAR RNA hairpin wer e shown to be thermodynamically stable and good substrates for binding by the HIV-1 Tat protein which normally bind to natural TAR (6). In t his study, we have extended our studies to the design, synthesis and a nalysis of the binding properties of covalently closed, double-strande d, cyclic RNA miniduplexes. A strategy using automated chemical synthe sis and T4 RNA ligase-catalyzed cyclization was employed to generate c yclic oligoribonucleotides. When both ends of a shortened, wild-type T AR RNA stem (9 bp) were covalently linked through either nucleotidic l oops (4 - 6 nt) or synthetic linkers (derivatized from hexaethylene gl ycol), the resulting cyclic TAR RNA analogs were good substrates for b inding by both Tat-derived peptide or full-length Tat protein. Interes tingly, the cyclic TAR analogs failed to show any binding if the synth etic linker was reduced in length (e.g. derivatized from triethylene g lycol), although such linkers are acceptable in the hairpin-shaped min iduplexes series (6). This implies that RNA conformational changes are required for Tat binding and that these changes are restricted in cer tain cyclic variants. Our findings suggest that covalently-closed nucl eic acid miniduplexes may be useful both to study nucleic acid-protein interactions as well as to provide a basis for therapeutic interventi on as transcription decoys.