Syntheses of RNAs with up to 100 Nucleotides Containing Site-Specific 2‘-Methylseleno Labels for Use in X-ray Crystallography

Abstract

The derivatization of nucleic acids with selenium is a new and highly promising approach to facilitate their three-dimensional structure determination by X-ray crystallography. Here, we report a comprehensive study on the chemical and enzymatic syntheses of RNAs containing 2'-methylseleno (2'-Se-methyl) nucleoside labels. Our approach includes the first synthesis of an appropriate purine nucleoside phosphoramidite building block. Most importantly, a substantially changed RNA solid-phase synthesis cycle, comprising treatment with threo-1,4-dimercapto-2,3-butanediol (DTT) after the oxidation step, is required for a reliable strand elongation. This novel operation allows for the chemical syntheses of multiple Se-labeled RNAs in sizes that can typically be achieved only for nonmodified RNAs. In combination with enzymatic ligation, biologically important RNA targets become accessible for crystallography. Exemplarily, this has been demonstrated for the Diels-Alder ribozyme and the add adenine riboswitch sequences. We point out that the approach documented here has been the chemical basis for the very recent structure determination of the Diels-Alder ribozyme which represents the first novel RNA fold that has been solved via its Se-derivatives.

A fast selenium derivatization strategy for crystallization and phasing of RNA structures

“…The 29-SeCH 3 -labeling method, suited for the determination of RNA structures, relies on the crystallization of RNA molecules that have natural nucleoside(s) substituted for their 29-SeCH 3 -modified counterparts during chemical RNA synthesis. The 29-SeCH 3 labeling allows use of the powerful multiwavelength anomalous dispersion (MAD) technique for structure determination and, in contrast to the halogenation of pyrimidines, can be utilized for the modification of all four standard nucleosides (adenosine, guanosine, uridine, and cytidine) (Hobartner and Micura 2004;Hobartner et al 2005;Moroder et al 2006;Puffer et al 2008). Moreover, the combination of the 29-SeCH 3 labeling with enzymatic ligation expands the z40 nucleotide (nt) limit of chemically synthesized Se-labeled RNA to large biologically relevant RNA molecules .…”

Structure determination and dynamics of protein–RNA complexes by NMR spectroscopy

“…A second approach described by Crothers and coworkers showed that sequence specific RNase H cleavage of an unlabeled and a labeled RNA can be followed by direct cross re-ligation of a labeled with an unlabeled fragment using T4 DNA ligase [145]. Finally, two groups presented another approach that combines the use of both T4 RNA and T4 DNA ligase in order to obtain multiple segmental isotopically labeled RNAs (i.e: three RNA fragments ligation) [139,146,147].…”

Crystal structure of an RNA polymerase ribozyme in complex with an antibody fragment

“…In addition to these problems, RNA molecules are prone to misfolding and adopting alternative conformations [11][12][13][14][15]. Moreover, once crystals are obtained, phasing for RNA crystals remains time-consuming and challenging compared with selenium-assisted phasing of protein crystals [11,16,17]. These problems have led RNA researchers to develop creative approaches to circumvent or ameliorate some of the problems.…”