Aptamers represent an emerging strategy to deliver cargo molecules, including dyes, drugs, proteins or even genes, into specific target cells. Upon binding to specific cell surface receptors aptamers can be internalized, for example by macropinocytosis or receptor mediated endocytosis. Here we report the in vitro selection and characterization of RNA aptamers with high affinity (Kd = 20 nM) and specificity for the human IL-6 receptor (IL-6R). Importantly, these aptamers trigger uptake without compromising the interaction of IL-6R with its natural ligands the cytokine IL-6 and glycoprotein 130 (gp130). We further optimized the aptamers to obtain a shortened, only 19-nt RNA oligonucleotide retaining all necessary characteristics for high affinity and selective recognition of IL-6R on cell surfaces. Upon incubation with IL-6R presenting cells this aptamer was rapidly internalized. Importantly, we could use our aptamer, to deliver bulky cargos, exemplified by fluorescently labeled streptavidin, into IL-6R presenting cells, thereby setting the stage for an aptamer-mediated escort of drug molecules to diseased cell populations or tissues.

The ability to rapidly identify small molecules that interact with RNA would have significant clinical and research applications. Low-molecular-weight molecules that bind to RNA have the potential to be used as drugs. Therefore, technologies facilitating the rapid and reliable identification of such activities become increasingly important. We have applied a fluorescence-based assay to screen for modulators of hammerhead ribozyme (HHR) catalysis from a small library of antibiotic compounds. Several unknown potent inhibitors of the hammerhead cleavage reaction were identified and further characterized. Tuberactinomycin A, for which positive cooperativity of inhibition in vitro was found, also reduced ribozyme cleavage in vivo. The assay is applicable to the screening of mixtures of compounds, as inhibitory activities were detected within a collection of 2,000 extracts from different actinomycete strains. This approach allows the rapid, reliable, and convenient identification and characterization of ribozyme modulators leading to insights difficult to obtain by classical methodology.

Background: Kunitz-type inhibitors provide a suitable scaffold for novel elastase inhibitors. Results: The inhibitor ShPI-1 was modified for pancreatic elastase binding, and the crystal structure of the complex was elucidated and analyzed. Conclusion: The extended protease-inhibitor interactions provide a potential switch to direct inhibitor selectivity toward elastases. Significance: These results will help to design novel elastase inhibitors for the treatment of tissue destruction diseases.

Low concentrations of the antibiotic doxycycline are sufficient to control the activity of an allosteric hammerhead ribozyme (shown schematically). This was obtained by allosteric selection of an RNA library, which was fused to helix II of the ribozyme. Such molecular switches may serve as a valuable tool for the development of tailored conditional gene expression systems that can be controlled by the presence or absence of any kind of small molecule.

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RNA-RNA interactions play a crucial role at many different levels of the cellular metabolism such as plasmid replication control, viral encapsidation, or transcriptional and translational regulation. Therefore, methods are necessary to investigate the molecular determinants of given interactions, including their stabilities, or to screen for new interacting partners. We designed an RNAhybrid system in S. cerevisiae, based on the yeast three-hybrid system. In this setup, the activation of a reporter gene is dependent on the interaction of two RNAs. A loop-loop interaction similar to the dimerization initiation site of the HIV genome was used as a model system, demonstrating that in this novel RNA-hybrid system only cognate RNAs promote the activation of the reporter gene. Levels of reporter activation correlate well with interaction stabilities determined in vitro by UV melting analyses, suggesting that conditions used for the analysis of in vitro structural stabilities translate well into the intracellular environment. Furthermore, the system was applicable for a screen against a test library. Nine out of ten selected clones were identified as predicted interaction partners for the bait RNA. In summary, we present a yeast reporter system depending on RNA-RNA interactions, which can be used alternatively for analysis of known interactions or for screening libraries in search for new interaction partners.

The crystal structure of a streptomycin binding RNA aptamer displays a novel bipartite fold able to clamp the antibiotic. In view of the recent findings that metabolites directly control mRNA translation, we might expect that similar structures exist in natural RNAs.