Fundamentos pedagógicos de educación a distancia

Among the most commonly utilized assays that have been developed to characterize RBP- RNA biochemical interactions in vitro are:

∙ Systematic evolution of ligands by exponential selection (SELEX) identifies high-affinity ligands for a protein of interest through sequential cycles of ligand selection from a pool of variant sequences and amplification of the bound sequences (Tuerk and Gold

[1990]). Multiple rounds of enrichment selection result in the exponential increase of high-affinity ligands, which can then be clonally isolated and characterized through electrophoresis and Sanger sequencing. While SELEX typically identifies one or a few consensus sequences of an RNA binding protein de novo, it is not quantitative and doesn’t provide information about an RBP’s lower affinity sites.

∙ The RNA electrophoretic mobility shift (EMSA) or ‘gel-shift’ assay allows for the rapid detection, visualization, and quantification of protein-RNA interactions. In a gel-shift experiment, unlabeled protein is incubated within vitro-generated RNA 5’ end-labeled with [𝛾-32_{P] ATP. Protein-RNA complexes are separated from unbound (free) RNA}

by native, nondenaturing polyacrylamide gel electrophoresis (PAGE). The amount of bound RNA in the complex as well as the free RNA is measured via phosphorimaging, with the fraction of bound RNA plotted as a function of protein concentration. From this curve, the apparent equilibrium binding constant (𝐾𝑑), defined as the concentra-

tion of protein at which 50% of the RNA is bound, can be derived as a measure of the affinity that the protein has for the particular RNA assayed. An advantage of the gel-shift assay is that it provides an absolute 𝐾𝑑 for the protein-RNA interaction,

though previous knowledge of a potential RNA substrate for the RBP of interest is required (Yakhnin et al. [2012]).

∙ Surface plasmon resonance (SPR) is a real-time, label-free optical biosensing technol- ogy that provides kinetic information about the rates of association and dissociation of an RBP for an RNA ligand of interest (Katsamba et al. [2002]). The RNA is im- mobilized to a gold sensor surface and a solution containing the RBP is flowed over

the surface while a light source shines on the sensor chip and is reflected to a detector. As the RBP solution is injected into the flow cell and binds to the RNA ligand, a change in the refractive index causes some of the light to be reflected at a different angle, with measurement of this index throughout RBP injection and wash out over the course of minutes at multiple different protein concentrations allowing inference of the association (𝑘𝐴) and dissociation rates (𝑘𝐷), and thus the dissociation constant

𝐾𝐷 = 𝑘_𝑘𝐷

𝐴. SPR was originally used to study two RRM-containing RBPs and mutants

and individual RBDs thereof (Katsamba et al. [2002]), and has more recently been utilized to provide absolute dissociation constants for RBFOX2 in a previous RNA Bind-n-Seq study (Lambert et al. [2014]). While SPR is a powerful method for mea- suring intermolecular interactions in real time, it requires specific instrumentation and expensive consumables, making it impractical for profiling hundreds of RBPs.

∙ In RNAcompete, a purified epitope-tagged RBP selects RNA sequences from an RNA pool of∼240,000 designed mostly unstructured sequences up to 41 nt in length. Bound RNAs are identified via microarray hybridization and the 7mer binding profile of an RBP of interest is determined computationally (Ray et al. [2017]). Originally applied to nine yeast and human RBPs (Ray et al. [2009]), it has subsequently been applied to 205 RBPs from 24 diverse eukaryotic species (Ray et al. [2013]), and a more re- cent adaptation using a sequencing-based approach produced “Sequence and Structure Models" (SSMs) derived from 40mers for seven yeast and human RBPs performed at a single protein concentration (RNAcompeteS, Cook et al.[2017]).

∙ In RNA Bind-n-Seq (RBNS), a purified epitope-tagged RBP (consisting minimally of the RNA binding domains plus 50 flanking amino acids on the N- and C-terminal ends) is incubated with a pool of random 20 or 40 nt oligonucleotides, and the pulled down RBP-bound RNA is subjected to high-throughput sequencing (Lambert et al.[2014]). Typically five separate incubation reactions are performed with differing quantities of the tagged RBP (5 - 1300 nM), with each of these five libraries as well as the input RNA sequenced to a depth of∼15-20 million reads. Computational analysis of the pulldown reads compared to the input reads provides the full spectrum of bound motifs (including

high and moderate affinity RNA sequences) as well as their secondary structure and context preferences. Importantly, because the RBNS oligo pool is random in contrast to the designed pool of ∼250,000 oligos used in RNAcompete, the RBP is presented with motifs in a wide variety of sequence and secondary structure contexts, enabling the fine-tuned dissection of an RBP’s specificity and affinity landscape.

∙ Other in vitro techniques that have been used to profile the specificity and/or affinity of one or a few RBPs include: SEQRS (Selection, high-throughput sequencing of RNA andSSLs (sequence specificity landscapes),Campbell et al. [2012]); RNA-MaP (quan- titative analysis of RNA on aMassivelyParallel array,Buenrostro et al.[2014]); HiTS- RAP (High-ThroughputSequencing -RNAAffinityProfiling,Tome et al.[2014]); and RNA-MITOMI (RNA-Mechanically Induced Trapping Of Molecular Interactions,

Martin et al. [2012]).