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Dissecting RNA folding by nucleotide analog interference mapping (NAIM)

Nature Protocols volume 3, pages 811823 (2008) | Download Citation

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Abstract

Nucleotide analog interference mapping (NAIM) is a powerful chemogenetic approach that allows RNA structure and function to be characterized at the atomic level. Random modifications of base or backbone moieties are incorporated into the RNA transcript as nucleotide analog phosphorothioates. The resulting RNA pool is then subjected to a stringent selection step, in which the RNA has to accomplish a specific task, for example, folding. RNA functional groups important for this process can be identified by physical isolation of the functional and the nonfunctional RNA molecules and subsequent mapping of the modified nucleotide positions in both RNA populations by iodine cleavage of the susceptible phosphorothioate linkage. This approach has been used to analyze a variety of aspects of RNA biochemistry, including RNA structure, catalysis and ligand interaction. Here, I describe how to set up a NAIM assay for studying RNA folding. This protocol can be readily adapted to study any RNAs and their properties. The time required to complete the experiment is dependent on the length of the RNA and the number of atomic modifications tested. In general, a single NAIM experiment can be completed in 1–2 weeks, but expect a time frame of several weeks to obtain reliable and statistically meaningful results.

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Acknowledgements

Anna Marie Pyle is acknowledged for her support and helpful suggestions. I thank Olga Fedorova for many invaluable discussions and for critically reading the manuscript. Funding was in part provided by National Institutes of Health (NIH) grant GM50313 to A.M. Pyle when I was a postdoctoral associate in her lab and by the Austrian Science Foundation (FWF; J2332) to C.W.

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  1. Max F. Perutz Laboratories, Department of Biochemistry, University of Vienna, Dr. Bohrgasse 9/5, Vienna 1030, Austria.

    • Christina Waldsich

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Correspondence to Christina Waldsich.

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    Supplementary Table 1

    Sample NAIM data

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https://doi.org/10.1038/nprot.2008.45

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