This application describes general strategies for the recognition of structured RNAs, and the continued development of methodology for the mapping of higher-order RNA structure. The ability to design molecules that bind discrete RNAs with high affinity and specificity has major implications for the treatment of oncogenic and viral disease. The ability to determine experimentally the tertiary fold of an RNA molecule is a critical first step in the design of inhibitors active at the RNA level. This application focuses on the recognition, higher-order structure, and protein-binding of the Rev Response Element (RRE), a 234 nucleotide RNA located within the env coding region of the HIV-1 genome. The RRE interacts with the viral protein Rev in the nucleus of infected cells to regulate the cytoplasmic appearance of mRNAs encoding viral structural proteins. The RRE is a clear target for antiviral therapy as well as fundamental research on the interactions of RNAs with proteins, with ligands, and with themselves. A new class of molecules (Tethered Oligonucleotide Probes, TOPs) constructed during the last period bind RNAs on the basis of both sequence and tertiary structure. TOPs consist of two short oligonucleotides linked by a flexible, synthetic tether. Here is proposed an in vitro selection experiment to screen DNA and RNA """"""""sequence space"""""""" for nucleic acid tethers that replace the synthetic tether and increase the stability of the TOP:RRE complex, the rate of its formation, or both. Such molecules should be preorganized for binding, or interact with the RRE through tertiary interactions. The proposed research may lead to the identification of DNA and RNA sequences that expand the nucleic acid structural repertoire, and could lead to new strategies for antisense inhibitors. A second strategy for accommodating RNA structure is proposed in which a TOP targets a single- and a double-stranded region; the single- stranded region via duplex formation and the double-stranded region via triplex formation. With methodology developed during the last period, molecules will be synthesized that contain the RNA cleavage agent EDTA.Fe(II) joined to a single, internal site within the RRE. Incubation of the modified RREs with a reductant will induce RNA self-cleavage at sites close in tertiary space to the attachment site. With these modified RREs, we will examine whether proposed base-pairing interactions within the stem II region are present in the free RRE or are induced by Rev binding, how these changes depend on Mg2+, how conformation within stem II changes when RRE binds Rev, and how conformation changes when RRE binds RBP9-27. An analogous experiment using structurally well-characterized tRNA(phe) will be performed to establish guidelines for data interpretation. The prediction of RNA structure from sequence is impeded by a lack of understanding of tertiary interactions. In addition to furthering our understanding of protein:RNA interactions, this research should allow identification of tertiary interactions that stabilize the RRE.
| Moses, A C; Huang, S W; Schepartz, A (1997) Inhibition of Rev.RRE complexation by triplex tethered oligonucleotide probes. Bioorg Med Chem 5:1123-9 |
