title: Pinpointing RNA-Protein Cross-Links with Site-Specific Stable Isotope-Labeled Oligonucleotides.
abstract: High affinity RNA-protein interactions are critical to cellular function, but directly identifying the determinants of binding within these complexes is often difficult. Here, we introduce a stable isotope mass labeling technique to assign specific interacting nucleotides in an oligonucleotide-protein complex by photo-cross-linking. The method relies on generating site-specific oxygen-18-labeled phosphodiester linkages in oligonucleotides, such that covalent peptide-oligonucleotide cross-link sites arising from ultraviolet irradiation can be assigned to specific sequence positions in both RNA and protein simultaneously by mass spectrometry. Using Lin28A and a let-7 pre-element RNA, we demonstrate that mass labeling permits unambiguous identification of the cross-linked sequence positions in the RNA-protein complex.
authors.affiliation: Department of Molecular Biology and Center for Computational and Integrative Biology, Howard Hughes Medical Institute, Massachusetts General Hospital , Boston, Massachusetts 02114, United States.
authors.forename: Victor S
authors.initials: VS
authors.lastname: Lelyveld
authors.affiliation: Department of Molecular Biology and Center for Computational and Integrative Biology, Howard Hughes Medical Institute, Massachusetts General Hospital , Boston, Massachusetts 02114, United States.|Department of Biosciences, Åbo Akademi University , Åbo FI-20520, Finland.
authors.forename: Anders
authors.initials: A
authors.lastname: Björkbom
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Ransey
authors.forename: Piotr
authors.initials: P
authors.lastname: Sliz
authors.affiliation: Department of Molecular Biology and Center for Computational and Integrative Biology, Howard Hughes Medical Institute, Massachusetts General Hospital , Boston, Massachusetts 02114, United States.|Department of Chemistry and Chemical Biology, Harvard University , Cambridge, Massachusetts 02138, United States.
authors.forename: Jack W
authors.initials: JW
authors.lastname: Szostak
mesh_terms: D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D012313:RNA / Q000737:chemistry*; D020871:RNA Stability
chemical_list: D003432:Cross-Linking Reagents; D009841:Oligonucleotides; D011506:Proteins; D012313:RNA