title: Mapping specificity landscapes of RNA-protein interactions by high throughput sequencing.
abstract: To function in a biological setting, RNA binding proteins (RBPs) have to discriminate between alternative binding sites in RNAs. This discrimination can occur in the ground state of an RNA-protein binding reaction, in its transition state, or in both. The extent by which RBPs discriminate at these reaction states defines RBP specificity landscapes. Here, we describe the HiTS-Kin and HiTS-EQ techniques, which combine kinetic and equilibrium binding experiments with high throughput sequencing to quantitatively assess substrate discrimination for large numbers of substrate variants at ground and transition states of RNA-protein binding reactions. We discuss experimental design, practical considerations and data analysis and outline how a combination of HiTS-Kin and HiTS-EQ allows the mapping of RBP specificity landscapes.
authors.affiliation: Center for RNA Molecular Biology, School of Medicine, Case Western Reserve University, 1099 Euclid Ave Cleveland, OH 44106, United States; Department of Biochemistry, School of Medicine, Case Western Reserve University, 1099 Euclid Ave Cleveland, OH 44106, United States. Electronic address: exj13@case.edu.
authors.forename: Eckhard
authors.initials: E
authors.lastname: Jankowsky
authors.affiliation: Department of Biochemistry, School of Medicine, Case Western Reserve University, 1099 Euclid Ave Cleveland, OH 44106, United States.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Harris
mesh_terms: D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D007700:Kinetics; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012343:RNA, Transfer / Q000235:genetics* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D043262:Ribonuclease P / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D016601:RNA-Binding Proteins; D014764:Viral Proteins; D012343:RNA, Transfer; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D043262:Ribonuclease P
keywords: Kinetics; Next generation sequencing; RBP; Thermodynamics