title: A microbead-based system for identifying and characterizing RNA-protein interactions by flow cytometry.
abstract: We present a high throughput, versatile approach to identify RNA-protein interactions and to determine nucleotides important for specific protein binding. In this approach, oligonucleotides are coupled to microbeads and hybridized to RNA-protein complexes. The presence or absence of RNA and/or protein fluorescence indicates the formation of an oligo-RNA-protein complex on each bead. The observed fluorescence is specific for both the hybridization and the RNA-protein interaction. We find that the method can discriminate noncomplementary and mismatch sequences. The observed fluorescence reflects the affinity and specificity of the RNA-protein interaction. In addition, the fluorescence patterns footprint the protein recognition site to determine nucleotides important for protein binding. The system was developed with the human protein U1A binding to RNAs derived from U1 snRNA but can also detect RNA-protein interactions in total RNA backgrounds. We propose that this strategy, in combination with emerging coded bead systems, can identify RNAs and RNA sequences important for interacting with RNA-binding proteins on genomic scales.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School and The Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA. alex_brodsky@dfci.harvard.edu
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Brodsky
authors.forename: Pamela A
authors.initials: PA
authors.lastname: Silver
mesh_terms: D001665:Binding Sites; D005434:Flow Cytometry / Q000379:methods*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008863:Microspheres; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D012334:RNA, Neoplasm / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000032:analysis*; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism*; D019809:Streptavidin / Q000737:chemistry
chemical_list: D007202:Indicators and Reagents; D008164:Luminescent Proteins; D009841:Oligonucleotides; D012334:RNA, Neoplasm; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; C405483:U1 small nuclear RNA; C075339:U1A protein; D049452:Green Fluorescent Proteins; D019809:Streptavidin