title: Comprehensive analysis of RNA-protein interactions by high-throughput sequencing-RNA affinity profiling.
abstract: RNA-protein interactions play critical roles in gene regulation, but methods to quantitatively analyze these interactions at a large scale are lacking. We have developed a high-throughput sequencing-RNA affinity profiling (HiTS-RAP) assay by adapting a high-throughput DNA sequencer to quantify the binding of fluorescently labeled protein to millions of RNAs anchored to sequenced cDNA templates. Using HiTS-RAP, we measured the affinity of mutagenized libraries of GFP-binding and NELF-E-binding aptamers to their respective targets and identified critical regions of interaction. Mutations additively affected the affinity of the NELF-E-binding aptamer, whose interaction depended mainly on a single-stranded RNA motif, but not that of the GFP aptamer, whose interaction depended primarily on secondary structure.
authors.affiliation: 1] Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA. [2].
authors.forename: Jacob M
authors.identifier: 0000000151072766
authors.initials: JM
authors.lastname: Tome
authors.affiliation: 1] Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA. [2].
authors.forename: Abdullah
authors.initials: A
authors.lastname: Ozer
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA.
authors.forename: John M
authors.initials: JM
authors.lastname: Pagano
authors.affiliation: Illumina, Inc., San Diego, California, USA.
authors.forename: Dan
authors.initials: D
authors.lastname: Gheba
authors.affiliation: Illumina, Inc., San Diego, California, USA.
authors.forename: Gary P
authors.initials: GP
authors.lastname: Schroth
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA.
authors.forename: John T
authors.initials: JT
authors.lastname: Lis
mesh_terms: D052157:Aptamers, Nucleotide; D020869:Gene Expression Profiling; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D059014:High-Throughput Nucleotide Sequencing; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*
chemical_list: D052157:Aptamers, Nucleotide; D011506:Proteins; D049452:Green Fluorescent Proteins; D012313:RNA