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Nat Methods. 2015 Sep;12(9):866-72. doi: 10.1038/nmeth.3490. Epub 2015 Aug 3.

Mutational interference mapping experiment (MIME) for studying RNA structure and function.

Author information

1
Architecture et Réactivité de l'ARN, Institut de Biologie Moléculaire et Cellulaire du Centre National de la Recherche Scientifique, Université de Strasbourg, Strasbourg, France.
2
BGI-Tech, BGI-Shenzhen, Shenzhen, China.
3
Centre for Virology, Burnet Institute, Melbourne, Victoria, Australia.
4
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
5
Faculty of Health, School of Medicine, Deakin University, Geelong, Victoria, Australia.
6
Commonwealth Scientific and Industrial Research Organization, Australian Animal Health Laboratory, Geelong, Victoria, Australia.
7
Department of Mathematics and Computer Science, Freie Universität, Berlin, Germany.

Abstract

RNA regulates many biological processes; however, identifying functional RNA sequences and structures is complex and time-consuming. We introduce a method, mutational interference mapping experiment (MIME), to identify, at single-nucleotide resolution, the primary sequence and secondary structures of an RNA molecule that are crucial for its function. MIME is based on random mutagenesis of the RNA target followed by functional selection and next-generation sequencing. Our analytical approach allows the recovery of quantitative binding parameters and permits the identification of base-pairing partners directly from the sequencing data. We used this method to map the binding site of the human immunodeficiency virus-1 (HIV-1) Pr55(Gag) protein on the viral genomic RNA in vitro, and showed that, by analyzing permitted base-pairing patterns, we could model RNA structure motifs that are crucial for protein binding.

PMID:
26237229
DOI:
10.1038/nmeth.3490
[Indexed for MEDLINE]

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