SELEX targeting mRNAs: The hunt for novel riboregulators (Immune deficiency)

There exists always a great deal of demand for novel medical therapies. With the continuing emergence of drug-resistant and entirely novel strains of bacteria and viruses, it is critical that researchers respond quickly to exploit the potential afforded by advances in the understanding of cellular processes. One such cellular regulatory process known as riboregulation came to light recently. Riboregulation involves the regulation of cellular events via RNA-RNA interactions. Riboregulators such as E. coli DsrR RNA globally regulate gene expression at the level of translation. In this work, we endeavored to determine whether novel non-antisense aptamer RNAs, obtained via in vitro selection experiments against mRNA elements, could be identified and shown to have appreciable effects on translation of the mRNAs. A subset of such RNAs could represent novel riboregulators. We established the concept by conducting SELEX studies in which we targeted the HIV-1 gag-pol frameshift site. We recovered RNAs that both inhibited frameshift-dependent translation and that positively affected both normal and frameshift-dependent translation. We then attempted to determine whether translation-stimulating aptamers could be recovered from SELEX experiments targeting an entirely unrelated mRNA target. As a target for in vitro selection, we chose a stem-loop near the start codon of A. thaliana epsps mRNA. We recovered a consensus translation-stimulating aptamer and attempted to determine if it was possible to express this aptamer RNA at a high level in A. thaliana plants. Using the powerful 35S plant promoter, we expressed mRNA-binding aptamers in transgenic plants and evaluated their in vivo function. Taqman RTPCR experiments confirmed the expression of aptamer RNAs at an estimated level (between 0.25--1% of total mRNA) comparable to what has been observed previously with the 35S constitutive promoter.