Nucleobase-Modified Triplex-Forming Peptide Nucleic Acids for Sequence-Specific Recognition of Double-Stranded RNA

Because of the important roles noncoding RNAs play in gene expression, their sequence-specific recognition is important for both fundamental science and the pharmaceutical industry. However, most noncoding RNAs fold in complex helical structures that are challenging problems for molecular recognition. Herein, we describe a method for sequence-specific recognition of double-stranded RNA using peptide nucleic acids (PNAs) that form triple helices in the major grove of RNA under physiologically relevant conditions. We also outline methods for solid-phase conjugation of PNA with cell-penetrating peptides and fluorescent dyes. Protocols for PNA preparation and binding studies using isothermal titration calorimetry are described in detail

High-throughput micro-characterization of RNA-protein interactions

12 p.-2 fig.-2 tabMany cellular processes depend on and are regulated by nucleic acid-protein interactions. In particular, RNA-binding proteins (RBPs) are involved in transcription, translation, modulating RNA polymerase activity, and stabilizing protein-RNA complexes. Furthermore, RBPs participate in the development of pathologies such as cancer and viral infections, and their dysfunction leads to mutations and the aberrant expression of noncoding RNAs. Therefore, the study of RNA-protein interactions represents a central issue for biology and biomedicine. While many valuable insights have been obtained from electrophoretic mobility shift assays (EMSA) and immunoprecipitation (IP), these standard methods suffer from two main limitations: insufficient sensitivity to capture low concentration RBP-RNA complexes in vitro and identification of interactions in vivo. In recent years, high-throughput (HTP) platforms have emerged that combine methodological improvements over conventional techniques with more sensitive detection systems, thereby catalyzing the simultaneous probing and analysis of a vast amount of RBP-RNA interactions by cellular proteomics and interactomics approaches. In this chapter, we summarize a selection of state-of-the-art in vitro, in vivo, and computational HTP platforms for the discovery and characterization of RNA-protein interactions. We also reflect on the wealth of information obtained by the structural analysis of RBPs and their RNA-binding domains as a valuable resource for the rational design and implementation of new RNA-binding discovery platforms.MCV has received funding from the Spanish Ministerio de Economía y Competitividad (CTQ2015-66206-C2-2-R and SAF2015-72961-EXP) and the Regional Government of Madrid (S2017/BMD-3673).Peer reviewe