Nucleic Acid Crystallography via Direct Selenium Derivatization: RNAs Modified with Se-Nucleobases

Abstract

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).

Theoretical characterization of sulfur-to-selenium substitution in an emissive RNA alphabet: impact on H-bonding potential and photophysical properties

“…The usage of a selenium atom in nucleic acid bases has already been reported. [29][30][31][32] The replacement of sulfur in tz-bases with selenium in ts-bases may possess two possible advantages, the former one being that the selenium in ts-bases could act as a scattering label for the phase determination in X-ray crystallography, the latter one being the enhanced absorption and emission when ts-bases are used instead (discussed below, in Section 3.6). Our previous computational investigation of tz-bases has clearly pointed out the minimal impact of these bases on the H-bonding and the energetics of the base pairs they are involved in ref.…”

“…The basis for such a substitution is in the fact that selenium is well known to feature anomalous diffraction in X-ray crystallography, thus helping to solve the phase problem. 26,27 Furthermore, selenium is present in RNA natural posttranscriptional modifications 28 and has already been artificially introduced in nucleic acid bases, in place of an oxygen atom for solving the phase problem, 29 but also for changing their pairing specificity 30,31 Notably, these two effects have been combined in a recent study proposing a Se-based modification. 32 The nucleobases composing this new fluorescent alphabet are shown in Fig.…”