Three-dimensional Structure of a Kunitz-type Inhibitor in Complex with an Elastase-like Enzyme.

Elastase-like enzymes are involved in important diseases such as acute pancreatitis, chronic inflammatory lung diseases, and cancer. Structural insights into their interaction with specific inhibitors will contribute to the development of novel anti-elastase compounds that resist rapid oxidation and proteolysis. Proteinaceous Kunitz-type inhibitors homologous to the bovine pancreatic trypsin inhibitor (BPTI) provide a suitable scaffold, but the structural aspects of their interaction with elastase-like enzymes have not been elucidated.

The small RNA RybA regulates key-genes in the biosynthesis of aromatic amino acids under peroxide stress in E. coli.

In bacteria, adaptive response to external stimuli is often regulated by small RNAs (sRNAs). In Escherichia coli, the organism in which sRNAs have been best characterized so far, no function could be attributed to 40 out of 79 sRNAs. Here we decipher the function of RybA, one of these orphan sRNAs.

Selecting allosteric ribozymes.

Allosteric ribozymes can be designed to respond to virtually any molecule of choice. The resulting species may be used for example as synthetic regulators of gene expression or alternatively as biosensors. In vitro selection techniques allow the isolation of active molecules from libraries as large as 10(15) different molecules.

Interleukin-6 receptor specific RNA aptamers for cargo delivery into target cells.

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).

In vitro selection of allosteric ribozymes.

In vitro selection techniques offer powerful and versatile methods to isolate nucleic acid sequences with specific activities from huge libraries. The present protocol describes an in vitro selection strategy for the de novo selection of allosteric self-cleaving ribozymes responding to virtually any drug of choice. We applied this method to select hammerhead ribozymes inhibited specifically by doxycycline or pefloxacin in the sub-micromolar range.

Identification and detection of RNA-RNA interactions using the yeast RNA hybrid system.

To test RNA-RNA interactions in cells, we developed a yeast RNA hybrid system derived from the yeast three-hybrid system. In this setup, the activation of a reporter gene (HIS3 or lacZ) is dependent on the interaction of two RNAs. One ('RNA X') is fused to MS2 RNA, forming the bait, which binds to a fusion protein composed of the MS2 coat and the LexA proteins.

Stabilities of HIV-1 DIS type RNA loop-loop interactions in vitro and in vivo.

RNA loop-loop interactions are a prevalent motif in the formation of tertiary structure and are well suited to trigger molecular recognition between RNA molecules. We determined the stabilities of several loop-loop interactions with a constant 6 bp core sequence and varying unpaired flanking nucleotides and found that the flanking bases have a strong influence on the stability and ion dependence of the kissing complex. In general, the stabilities determined in 1 M Na+ are equivalent to those in the presence of near physiological Mg2+ concentrations.

A yeast RNA-hybrid system for the detection of RNA-RNA interactions in vivo.

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 RNA-hybrid system in S.

Aptamer structures: a preview into regulatory pathways?

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.