Redirection of miRNA-Argonaute Complexes to Specific Target Sites by Synthetic Adaptor Molecules.

Dysregulation of miRNAs is connected with a multitude of diseases for which antagomirs and miRNA replacement are discussed as therapeutic options. Here, we suggest an alternative concept based on the redirection of RISCs to non-native target sites. Metabolically stable DNA-LNA mixmers are used to mediate the binding of RISCs to mRNAs without any direct base complementarity to the presented guide RNA strand. Physical redirection of a dye-labeled miRNA model and of specific miRNA-programmed RISC fractions present in HeLa extracts is demonstrated by pull-down experiments with biotinylated capture oligonucleotides.

Peptidomimetics That Inhibit and Partially Reverse the Aggregation of Aß1-42.

The peptide sequence KLVFF resembles the hydrophobic core of the Aß peptide known to form amyloid plaques in Alzheimer's disease. Starting from its retro-inverso peptide, we have synthesized three generations of peptidomimetics. Step by step natural amino acids have been replaced by aromatic building blocks accessible from the Pd-catalyzed Catellani reaction. The final compound 18 is stable against proteolytic decay and largely prevents the aggregation of Aß1-42 over extended periods of time. The activity of the new inhibitors was tested first by fluorescence correlation spectroscopy. For closer examination of compound 18, additional techniques were also applied: laser-induced liquid bead ion desorption mass spectrometry, confocal laser scanning microscopy, thioflavin T fluorescence, and gel electrophoresis. Compound 18 not only retards the aggregation of chemically synthesized Aß but also can partially dissolve the oligomeric structures. Thioflavin binding mature fibrils, however, seem to resist the inhibitor.

Sequence-specific RNA cleavage by PNA conjugates of the metal-free artificial ribonuclease tris(2-aminobenzimidazole).

Tris(2-aminobenzimidazole) conjugates with antisense oligonucleotides are effective site-specific RNA cleavers. Their mechanism of action is independent of metal ions. Here we investigate conjugates with peptide nucleic acids (PNA). RNA degradation occurs with similar rates and substrate specificities as in experiments with DNA conjugates we performed earlier. Although aggregation phenomena are observed in some cases, proper substrate recognition is not compromised. While our previous synthesis of 2-aminobenzimidazoles required an HgO induced cyclization step, a mercury free variant is described herein.

Fragment based search for small molecule inhibitors of HIV-1 Tat-TAR.

Basic molecular building blocks such as benzene rings, amidines, guanidines, and amino groups have been combined in a systematic way to generate ligand candidates for HIV-1 TAR RNA. Ranking of the resulting compounds was achieved in a fluorimetric Tat-TAR competition assay. Although simple molecules such as phenylguanidine are inactive, few iteration steps led to a set of ligands with IC50 values ranging from 40 to 150 µM. 1,7-Diaminoisoquinoline 17 and 2,4,6-triaminoquinazoline 22 have been further characterized by NMR titrations with TAR RNA. Compound 22 is bound to TAR at two high affinity sites and shows slow exchange between the free ligand and the RNA complex. These results encourage investigations of dimeric ligands built from two copies of compound 22 or related heterocycles.

Laser-induced liquid bead ion desorption mass spectrometry: an approach to precisely monitor the oligomerization of the ß-amyloid peptide.

In the present work, the recently developed laser-induced liquid bead ion desorption mass spectrometry (LILBID MS) is applied as a novel technique to study Aß oligomerization, thought to be crucial in Alzheimer's disease (AD). The characterization of the earliest nucleation events of this peptide necessitates the application of several techniques to bridge the gap between small oligomers and large fibrils. We precisely monitored in time the transformation of monomeric Aß (1-42) into oligomeric Aß(n) (n < 20) and its dependence on concentration and agitation. The distribution shows signs of the hexamer being crucial in the assembly process. The intensity of the monomer decreases in time with a time constant of about 9 h. After a lag time of around 10 h, a phase transition occurred in which the total ion current of the oligomers goes to nearly zero. In this late stage of aggregation, protofibrils are formed and mass spectrometry is no longer sensitive. Here fluorescence correlation spectroscopy (FCS) and transmission electron microscopy (TEM) are complementary tools for detection and size characterization of these large species. We also utilized the oligomers of Aß (1-42) as a model of the corresponding in vivo process to screen the efficacy and specificity of small molecule inhibitors of oligomerization. The LILBID results are in excellent agreement with condensed phase behavior determined in other studies. Our data identified LILBID MS as a powerful technique that will advance the understanding of peptide oligomerization in neurodegenerative diseases and represents a powerful tool for the identification of small oligomerization inhibitors.

Metal-free catalysts for the hydrolysis of RNA derived from guanidines, 2-aminopyridines, and 2-aminobenzimidazoles.

2-aminopyridine and 2-aminobenzimidazole were chosen as structural analogues to substitute guanidinium groups in receptor molecules designed as phosphoryl transfer catalysts. Shifting the pKa of the guanidinium analogues toward 7 was expected to raise catalytic activities in aqueous buffer. Although the pKa's of both heterocycles are similar (6.2 and 7.0), only 2-aminobenzimidazole led to active RNA cleavers. All cleavage assays were run with fluorescently labeled substrates and a DNA sequencer. RNase contaminations would degrade RNA enantioselectively. In contrast, achiral catalysts such as 9b and 10b necessarily induce identical cleavage patterns in RNA and its mirror image. This principle allowed us to safely rule out contamination effects in this study. The most active catalysts, tris(2-aminobenzimidazoles) 9b and 10b, were shown by fluorescence correlation spectroscopy (FCS) to aggregate with oligonucleotides. However, at very low concentrations the compounds are still active in the nonaggregated state. Conjugates of 10b with antisense oligonucleotides or RNA binding peptides, therefore, will be promising candidates as site specific artificial ribonucleases.