The cellular functions of RNA are not limited to their role as blueprints for protein synthesis. In particular, noncoding RNA, such as, snRNAs, lncRNAs, miRNAs, play important roles. With increasing numbers of RNAs being identified, it is well known that the transcriptome outnumbers the proteome by far. This emphasizes the great importance of functional RNA characterization and the need to further develop tools for these investigations, many of which are still in their infancy. Fluorescent light-up aptamers (FLAPs) are RNA sequences that can bind nontoxic, cell-permeable small-molecule fluorogens and enhance their fluorescence over many orders of magnitude upon binding. FLAPs can be encoded on the DNA level using standard molecular biology tools and are subsequently transcribed into RNA by the cellular machinery, so that they can be used as fluorescent RNA tags (FLAP-tags). In this Minireview, we give a brief overview of the fluorogens that have been developed and their binding RNA aptamers, with a special focus on published crystal structures. A summary of current and future cellular FLAP applications with an emphasis on the study of RNA-RNA and RNA-protein interactions using split-FLAP and Förster resonance energy transfer (FRET) systems is given.

Structural information about protein-RNA complexes supports the understanding of crucial recognition processes in the cell, and it can allow the development of high affinity ligands to interfere with these processes. In this respect, the identification of amino acid hotspots is particularly important. In contrast to protein-protein interactions, in silico approaches for protein-RNA interactions lag behind in their development. Herein, we report an analysis of available protein-RNA structures. We assembled a data set of 322 crystal and NMR structures and analyzed them regarding interface properties. In addition, we describe a computational alanine-scanning approach which provides interaction scores for interface amino acids, allowing the identification of potential hotspots in protein-RNA interfaces. We have made the computational approach available as an online tool, which allows interaction scores to be calculated for any structure of a protein-RNA complex by uploading atomic coordinates to the PRI HotScore web server (https://prihotscore.labs.vu.nl).

The design of high-affinity, RNA-binding ligands has proven very challenging. This is due to the unique structural properties of RNA, often characterized by polar surfaces and high flexibility. In addition, the frequent lack of well-defined binding pockets complicates the development of small molecule binders. This has triggered the search for alternative scaffolds of intermediate size. Among these, peptide-derived molecules represent appealing entities as they can mimic structural features also present in RNA-binding proteins. However, the application of peptidic RNA-targeting ligands is hampered by a lack of design principles and their inherently low bio-stability. Here, the structure-based design of constrained α-helical peptides derived from the viral suppressor of RNA silencing, TAV2b, is described. We observe that the introduction of two inter-side chain crosslinks provides peptides with increased α-helicity and protease stability. One of these modified peptides (B3) shows high affinity for double-stranded RNA structures including a palindromic siRNA as well as microRNA-21 and its precursor pre-miR-21. Notably, B3 binding to pre-miR-21 inhibits Dicer processing in a biochemical assay. As a further characteristic this peptide also exhibits cellular entry. Our findings show that constrained peptides can efficiently mimic RNA-binding proteins rendering them potentially useful for the design of bioactive RNA-targeting ligands.

The cellular functions of RNA are not limited to their role as blueprints for protein synthesis. In particular, noncoding RNA, such as, snRNAs, lncRNAs, miRNAs, play important roles. With increasing numbers of RNAs being identified, it is well known that the transcriptome outnumbers the proteome by far. This emphasizes the great importance of functional RNA characterization and the need to further develop tools for these investigations, many of which are still in their infancy. Fluorescent light‐up aptamers (FLAPs) are RNA sequences that can bind nontoxic, cell‐permeable small‐molecule fluorogens and enhance their fluorescence over many orders of magnitude upon binding. FLAPs can be encoded on the DNA level using standard molecular biology tools and are subsequently transcribed into RNA by the cellular machinery, so that they can be used as fluorescent RNA tags (FLAP‐tags). In this Minireview, we give a brief overview of the fluorogens that have been developed and their binding RNA aptamers, with a special focus on published crystal structures. A summary of current and future cellular FLAP applications with an emphasis on the study of RNA–RNA and RNA–protein interactions using split‐FLAP and Förster resonance energy transfer (FRET) systems is given.

Roll with it: The quantitative analysis of specific miRNAs from biological samples is very likely to revolutionize diagnostics of human disease. A novel method for miRNA analysis employing rolling-circle amplification (RCA) can homogeneously detect miRNA, even at concentrations as low as 10 fM. The use of T4 RNA ligase 2 (T4 RnL2) at elevated temperatures enables very good discrimination of miRNAs differing by a single nucleotide.