Riboswitches are conserved functional domains in mRNA that mostly exist in bacteria. They regulate gene expression in response to varying concentrations of metabolites or metal ions. Recently, the NMT1 RNA motif has been identified to selectively bind xanthine and uric acid, respectively, both are involved in the metabolic pathway of purine degradation. Here, we report a crystal structure of this RNA bound to xanthine. Overall, the riboswitch exhibits a rod-like, continuously stacked fold composed of three stems and two internal junctions. The binding-pocket is determined by the highly conserved junctional sequence J1 between stem P1 and P2a, and engages a long-distance Watson–Crick base pair to junction J2. Xanthine inserts between a G–U pair from the major groove side and is sandwiched between base triples. Strikingly, a Mg2+ ion is inner-sphere coordinated to O6 of xanthine and a non-bridging oxygen of a backbone phosphate. Two further hydrated Mg2+ ions participate in extensive interactions between xanthine and the pocket. Our structure model is verified by ligand binding analysis to selected riboswitch mutants using isothermal titration calorimetry, and by fluorescence spectroscopic analysis of RNA folding using 2-aminopurine-modified variants. Together, our study highlights the principles of metal ion-mediated ligand recognition by the xanthine riboswitch.

The chemical synthesis of modified oligoribonucleotides represents a powerful approach to study the structure, stability, and biological activity of RNAs. Selected RNA modifications have been proven to enhance the drug-like properties of RNA oligomers providing the oligonucleotide-based therapeutic agents in the antisense and siRNA technologies. The important sites of RNA modification/functionalization are the nucleobase residues. Standard phosphoramidite RNA chemistry allows the site-specific incorporation of a large number of functional groups to the nucleobase structure if the building blocks are synthetically obtainable and stable under the conditions of oligonucleotide chemistry and work-up. Otherwise, the chemically modified RNAs are produced by post-synthetic oligoribonucleotide functionalization. This review highlights the post-synthetic RNA modification approach as a convenient and valuable method to introduce a wide variety of nucleobase modifications, including recently discovered native hypermodified functional groups, fluorescent dyes, photoreactive groups, disulfide crosslinks, and nitroxide spin labels.

A post-synthetic reaction of 5-pivaloyloxymethyluridine (present in a support-bound RNA oligomer) with various nucleophilic reagents furnished efficiently the corresponding products bearing one of the tRNA wobble 5-methyluridines (mnmU, cmnmU, τmU, nmU, inmU or cnmU). The syntheses of oligoribonucleotides modified with inmU and cnmU are reported for the first time.