A rapid and quantitative method to evaluate binding properties of hairpin RNAs to peptides using peptide microarrays has been developed. The microarray technology was shown to be a powerful tool for high-throughput analysis of RNA-peptide interactions by its application to profiling interactions between 111 peptides and six hairpin RNAs. The peptide microarrays were also employed to measure hundreds of dissociation constants (K(d)) of RNA-peptide complexes. Our results reveal that both hydrophobic and hydrophilic faces of amphiphilic peptides are likely involved in interactions with RNAs. Furthermore, these results also show that most of the tested peptides bind hairpin RNAs with submicromolar K(d) values. One of the peptides identified by using this method was found to have good inhibitory activity against TAR-Tat interactions in cells. Because of their great applicability to evaluation of nearly all types of RNA-peptide interactions, peptide microarrays are expected to serve as robust tools for rapid assessment of peptide-RNA interactions and development of peptide ligands against RNA targets.
Nuclear B-catenin forms a transcription complex with TCF-4, which is implicated in colon cancer development and progression. Recently, we and others have shown that B-catenin could be a regulator of RNA splicing and it also stabilizes the cyclooxygenase-2 (COX-2) mRNA. Here, we further explored the role of B-catenin in the RNA metabolism in colon cancer cells. To specifically modulate the subcellular functions of B-catenin, we expressed the RNA aptamer in the form of RNA intramers with unique cellular localizations. The nucleus-expressed RNA intramer proved to be effective in reducing the protein-protein interaction between B-catenin and TCF-4, thus shown to be a specific regulator of B-cateninactivated transcription. It could also regulate the alternative splicing of E1A minigene in diverse colon cancer cell lines. In addition, we tested whether B-catenin could stabilize any other mRNAs and found that cyclin D1 mRNA was also bound and stabilized by B-catenin. Significantly, the cytoplasmexpressed RNA intramer reverted the B-catenin-induced COX-2 and cyclin D1 mRNA stabilization. We show here that B-catenin regulated multiple steps of RNA metabolism in colon cancer cells and might be the protein factor coordinating RNA metabolism. We suggest that the RNA intramers could provide useful ways for inhibiting B-catenin-mediated transcription and RNA metabolism, which might further enhance the antitumorigenic effects of these molecules in colon cancer cells. [Cancer Res 2007;67(19):9315-21]
Doxorubicin is a widely used anti-cancer drug. It is assumed to act by inhibiting DNA replication or transcription, although its precise targets and mechanism of cytotoxicity remain unresolved. A T7 phage library expressing human liver cDNA was screened against immobilized doxorubicin to isolate doxorubicin binding proteins. The selected phage contained the C-terminal region of nucleolar phosphoprotein hNopp140, an important factor in the biogenesis of the nucleolus. When the cloned sequence was expressed in E. coli, the recombinant protein was phosphorylated by casein kinase II and oligomerized in the presence of magnesium and fluoride ions, as occurs in vivo. Doxorubicin bound to the expressed protein with a dissociation constant of 4.5 x 10(-6) M, and this interaction was inhibited by the phosphorylation of hNopp140. These results suggested that doxorubicin might disrupt the cellular function of hNopp140.
MicroRNA-155, one of the most potent miRNAs that suppress apoptosis in human cancer, is overexpressed in numerous cancers, and it displays oncogenic activity. Peptide microarrays, constructed by immobilizing 185 peptides containing the C-terminal hydrazide onto epoxide-derivatized glass slides, were employed to evaluate peptide binding properties of pre-miRNA-155 and to identify its binding peptides. Two peptides, which were identified based on the results of peptide microarray and in vitro Dicer inhibition studies, were found to inhibit generation of mature miRNA-155 catalyzed by Dicer and to enhance expression of miRNA-155 target genes in cells. In addition, the results of cell experiments indicate that peptide inhibitors promote apoptotic cell death via a caspase-dependent pathway. Finally, observations made in NMR and molecular modeling studies suggest that a peptide inhibitor preferentially binds to the upper bulge and apical stem-loop region of pre-miRNA-155, thereby suppressing Dicer-mediated miRNA-155 processing.
We describe the design and synthesis of new heterodimeric conjugates, which are comprised of a neomycin B (Neo) stem-binding component and a chloramphenicol (Cam) or linezolid (Lnz) loop-binding component. Some of the heterodimeric conjugates display enhanced affinities to RNA targets and that binding occurs in both stem and loop regions of the RNA. In addition, the results of foot-printing and mutation studies suggest that the enhanced binding affinity of the conjugates is RNA sequence-specific.
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