The 2′-hydroxyl group fulfills numerous structural and functional roles in RNA, including those of hydrogen bond donor and acceptor. While loss of function upon 2′-deoxynucleotide substitution establishes the importance of specific 2′-hydroxyl groups within RNA, this approach provides no information about how these hydroxyl groups impart their functional contribution. We use an atomic mutation cycle to evaluate the functional importance of the 2′-hydroxyl group's hydrogen atom. Using the Tetrahymena ribozyme reaction, we challenge the cycle to expose the catalytic contribution of the cleavage site 2′-hydroxyl group and its associated hydrogen bond network. The results establish the viability of this cycle as an approach to reveal 2′-hydroxyl groups that donate functionally significant hydrogen bonds.
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