Pinpointing RNA-Protein Cross-Links with Site-Specific Stable Isotope-Labeled Oligonucleotides.
Pinpointing RNA-Protein Cross-Links with Site-Specific Stable Isotope-Labeled Oligonucleotides.
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DOI：
10.1021/jacs.5b10596
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发表时间：
2015-12-16
影响因子：
15
通讯作者：
Szostak JW
中科院分区：
文献类型：
--
作者：
Lelyveld VS;Björkbom A;Ransey EM;Sliz P;Szostak JW
高亲和力的RNA-蛋白质相互作用对细胞功能至关重要，但直接鉴定这些复合物中的结合决定簇通常很困难。在这里，我们介绍了一种稳定的同位素质量标记技术，指定特定的相互作用的核苷酸在磷脂-蛋白质复合物的光交联。该方法依赖于在寡核苷酸中产生位点特异性氧-18-标记的磷酸二酯键，使得由紫外线照射产生的共价肽-寡核苷酸交联位点可以通过质谱法同时分配到RNA和蛋白质中的特定序列位置。使用Lin 28 A和let-7前元件RNA，我们证明了质量标记允许明确识别RNA-蛋白质复合物中的交联序列位置。
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.