Selections that optimize RNA display in the yeast three-hybrid system.

The yeast three-hybrid system (Y3H) is a powerful tool to select or confirm RNA-protein interactions. Target protein recognition of an RNA insert within a test transcript depends on at least three factors: intrinsic protein affinity for the properly folded insert, retention of RNA insert tertiary structure within a longer RNA transcript, and accessibility of the RNA insert to the target protein. Y3H reporter gene readout reflects the combination of these factors. Here, we discuss RNA insert tertiary structure and accessibility in the Y3H as "RNA display." We review evidence that RNA display can sometimes be optimized during Y3H selections that do not increase the intrinsic affinity of an RNA insert for a target protein. This situation is more likely when a library of RNA inserts and heterogeneous flanking sequences is subjected to selection, and is less likely when point mutations are targeted to the insert in a fixed context. An RNA display vector with enhanced modularity has been developed to minimize sequence context effects in the Y3H.

Characterization of anti-NF-kappaB RNA aptamer-binding specificity in vitro and in the yeast three-hybrid system.

RNA aptamers offer a potential therapeutic approach to the competitive inhibition of DNA-binding transcription factors. In previous reports we described in vitro selection and characterization of anti-NF-kappaB p50 and p65 RNA aptamers. We now describe the further characterization of these aptamers in vitro and in vivo. We show that sub-saturating concentrations of certain anti-p50 RNA aptamers promote complex formation with NF-kappaB p50 tetramers, whereas anti-p65 R1 RNA aptamers bind NF-kappaB dimers under all conditions tested. Yeast three-hybrid RNA aptamer specificity studies corroborate previous in vitro results, verifying that anti-p50 and anti-p65 R1 RNA aptamers are highly specific for NF-kappaB p50(2) and p65(2), respectively. These studies introduce a novel T-cassette RNA transcript that improves RNA display from a four-way RNA junction. Mutagenesis of the anti-p65 R1 aptamer reveals tolerated substitutions, suggesting a complex tertiary structure. We describe in vivo selections from a yeast three-hybrid RNA library containing sequences present early in the R1 SELEX process to identify novel anti-p65 RNA aptamers, termed Y1 and Y3. These aptamers appear to be compact bulged hairpins, reminiscent of anti-p50. Y1 competitively inhibits the DNA-binding domain of NF-kappaB p65(2) in vitro.

Selection and characterization of anti-NF-kappaB p65 RNA aptamers.

NF-kappaB transcription factors include a group of five mammalian proteins that form hetero- or homodimers and regulate hundreds of target genes involved in acute inflammation, HIV-1 transcription activation, and resistance to cancer therapy. We previously used in vitro selection to develop a small RNA aptamer (anti-p50) that binds the DNA-binding domain of NF-kappaB p50(2) with low nanomolar affinity but does not bind NF-kappaB p65(2). Here, we report the in vitro selection of anti-NF-kappaB p65 RNA aptamers using parallel in vitro selections with either a fully randomized RNA library or a degenerate RNA library based on the primary sequence of the 31-nucleotide anti-p50 RNA aptamer. We report the characterization of these aptamers with respect to NF-kappaB target specificity, affinity, minimal sequence requirements, secondary structure, and competition with DNA kappaB sites. These results expand opportunities for artificial inhibition of NF-kappaB transcription factor dimers containing p65 subunits.

Sequence-specific binding of DNA and RNA to immobilized Nickel ions.

Immobilization of divalent Nickel cations provides a tool for affinity purification of proteins containing hexahistidine tags. During experiments to generate single-stranded DNA aptamers to immobilized proteins we inadvertently identified DNA sequences with affinity for Nickel-nitrilotriacetic acid (Ni(2+)-NTA) magnetic beads. Analysis of these aptamers revealed that affinity for the Ni(2+)-NTA support requires only single-stranded sequences with multiple adenosine residues. Bound nucleic acids can be eluted with imidazole. A single-stranded dA(20) affinity tag (but not other homopolymer sequences) is sufficient for immobilization of double-stranded DNA PCR products on Ni(2+)-NTA magnetic beads. Addition of an rA(20) sequence to an RNA transcript allowed its affinity capture on Ni(2+)-NTA magnetic beads, suggesting an approach for purification of poly(A) mRNA.