Chemical probing of RNA with the hydroxyl radical at single-atom resolution
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Date
2014-11-10
DOI
Authors
Ingle, Shakti
Azad, Robert Navid
Jain, Swapan S.
Tullius, Thomas D.
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Published version
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Citation
Shakti Ingle, Robert N Azad, Swapan S Jain, Thomas D Tullius. 2014. "Chemical probing of RNA with the hydroxyl radical at single-atom resolution." Nucleic Acids Research, Volume 42, Issue 20, pp. 12758 - 12767 (10). https://doi.org/10.1093/nar/gku934
Abstract
While hydroxyl radical cleavage is widely used to map RNA tertiary structure, lack of mechanistic understanding of strand break formation limits the degree of structural insight that can be obtained from this experiment. Here, we determine how individual ribose hydrogens of sarcin/ricin loop RNA participate in strand cleavage. We find that substituting deuterium for hydrogen at a ribose 5′-carbon produces a kinetic isotope effect on cleavage; the major cleavage product is an RNA strand terminated by a 5′-aldehyde. We conclude that hydroxyl radical abstracts a 5′-hydrogen atom, leading to RNA strand cleavage. We used this approach to obtain structural information for a GUA base triple, a common tertiary structural feature of RNA. Cleavage at U exhibits a large 5′ deuterium kinetic isotope effect, a potential signature of a base triple. Others had noted a ribose-phosphate hydrogen bond involving the G 2′-OH and the U phosphate of the GUA triple, and suggested that this hydrogen bond contributes to backbone rigidity. Substituting deoxyguanosine for G, to eliminate this hydrogen bond, results in a substantial decrease in cleavage at G and U of the triple. We conclude that this hydrogen bond is a linchpin of backbone structure around the triple.
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© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.