Specificity of nucleobase pairing provides essential foundation for genetic information storage, replication, transcription and translation in all living organisms. However, the wobble base pairs, where U in RNA (or T in DNA) pairs with G instead of A, might compromise the high specificity of the base pairing. The U/G wobble pairing is ubiquitous in RNA, especially in non-coding RNA. In order to increase U/A pairing specificity, we have hypothesized to discriminate against U/G wobble pair by tailoring the steric and electronic effects at the 2-exo position of uridine and replacing the 2-exo oxygen with a selenium atom. We report here the first synthesis of the 2-Se-U-RNAs as well as the 2-Se-uridine (SeU) phosphoramidite. Our biophysical and structural studies of the SeU-RNAs indicate that this single atom replacement can indeed create a novel U/A base pair with higher specificity than the natural one. We reveal that the SeU/A pair maintains a structure virtually identical to the native U/A base pair, while discriminating against U/G wobble pair. This oxygen replacement with selenium offers a unique chemical strategy to enhance the base pairing specificity at the atomic level.
Selenium-derivatized RNAs are powerful tools for structure and function studies of RNAs and their protein complexes. By taking the advantage of selenium modifications, researchers can determine novel RNA structures via convenient SAD and MAD phasing. As one of the naturally occurring tRNA modifications, 2-seleno-uridine, which presents almost exclusively at the wobble position of anticodon loop in various bacterial tRNAs (Ching et al., Proc Natl Acad Sci U S A 82:347, 1985; Dunin-Horkawicz et al., Nucleic Acids Res 34:D145–D149, 2006), becomes one of the most promising modifications for crystallographic studies. Our previous studies have demonstrated many unique properties of 2-seleno-uridine, including stability (Sun et al., RNA 19:1309–1314, 2013), minimal structural perturbation (Sun et al., Nucleic Acids Res 40:5171–5179, 2012), and enhanced base-pairing fidelity (Sun et al., Nucleic Acids Res 40:5171–5179, 2012). In this protocol, we present the efficient chemical synthesis of 2-seleno-uridine triphosphate (SeUTP) and the facile transcription and purification of SeU-containing RNAs (SeU-RNA).
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