Chemical synthesis of the 5-taurinomethyl(-2-thio)uridine modified anticodon arm of the human mitochondrial tRNALeu(UUR) and tRNALys
Abstract
5-Taurinomethyluridine (τm5U) and 5-taurinomethyl-2-thiouridine (τm5s2U) are located at the wobble position of human mitochondrial (hmt) tRNALeu(UUR) and tRNALys, respectively. Both hypermodified units restrict decoding of the third codon letter to A and G. Pathogenic mutations in the genes encoding hmt-tRNALeu(UUR) and hmt-tRNALys are responsible for the loss of the discussed modifications and, as a consequence, for the occurrence of severe mitochondrial dysfunctions (MELAS, MERRF). Synthetic oligoribonucleotides bearing modified nucleosides are a versatile tool for studying mechanisms of genetic message translation and accompanying pathologies at nucleoside resolution. In this paper, we present site-specific chemical incorporation of τm5U and τm5s2U into 17-mers related to the sequence of the anticodon arms hmt-tRNALeu(UUR) and hmt-tRNALys, respectively employing phosphoramidite chemistry on CPG support. Selected protecting groups for the sulfonic acid (4-(tert-butyldiphenylsilanyloxy)-2,2-dimethylbutyl) and the exoamine function (-C(O)CF3) are compatible with the blockage of the canonical monomeric units. The synthesis of τm5s2U-modified RNA fragment was performed under conditions eliminating the formation of side products of 2-thiocarbonyl group oxidation and/or oxidative desulphurization. The structure of the final oligomers was confirmed by mass spectroscopy and enzymatic cleavage data.
Keywords
- human mitochondrial tRNA
- modified ribonucleosides
- 5-taurinomethyluridine
- 5-taurinomethyl-2-thiouridine
- phosphoramidite chemistry
Footnotes
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↵1 Corresponding author
E-mail grazyna.leszczynska{at}p.lodz.pl
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Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.044412.114.
- Received January 21, 2014.
- Accepted February 21, 2014.
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