| Autor: |
Ramanathan M; Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California, USA., Majzoub K; Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA., Rao DS; Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California, USA., Neela PH; Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California, USA., Zarnegar BJ; Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California, USA., Mondal S; Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California, USA., Roth JG; Department of Neurosurgery, Stanford University School of Medicine, Stanford, California, USA., Gai H; Department of Neurosurgery, Stanford University School of Medicine, Stanford, California, USA., Kovalski JR; Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California, USA., Siprashvili Z; Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California, USA., Palmer TD; Department of Neurosurgery, Stanford University School of Medicine, Stanford, California, USA., Carette JE; Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA., Khavari PA; Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California, USA.; Veterans Affairs Palo Alto Healthcare System, Palo Alto, California, USA. |
| Abstrakt: |
RNA-protein interactions play numerous roles in cellular function and disease. Here we describe RNA-protein interaction detection (RaPID), which uses proximity-dependent protein labeling, based on the BirA* biotin ligase, to rapidly identify the proteins that bind RNA sequences of interest in living cells. RaPID displays utility in multiple applications, including in evaluating protein binding to mutant RNA motifs in human genetic disorders, in uncovering potential post-transcriptional networks in breast cancer, and in discovering essential host proteins that interact with Zika virus RNA. To improve the BirA*-labeling component of RaPID, moreover, a new mutant BirA* was engineered from Bacillus subtilis, termed BASU, that enables >1,000-fold faster kinetics and >30-fold increased signal-to-noise ratio over the prior standard Escherichia coli BirA*, thereby enabling direct study of RNA-protein interactions in living cells on a timescale as short as 1 min. |
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