The Synthesis of Methylated, Phosphorylated, and Phosphonated 3'-Aminoacyl-tRNA super(Sec) Mimics

The twenty first amino acid, selenocysteine (Sec), is the only amino acid that is synthesized on its cognate transfer RNA (tRNA super(Sec)) in all domains of life. The multistep pathway involves O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase (SepSecS), an enzyme that catalyzes the terminal chemical reaction during which the phosphoseryl-tRNA super(Sec) intermediate is converted into selenocysteinyl-tRNA super(Sec). The SepSecS architecture and the mode of tRNA super(Sec) recognition have been recently determined at atomic resolution. The crystal structure provided valuable insights that gave rise to mechanistic proposals that could not be validated because of the lack of appropriate molecular probes. To further improve our understanding of the mechanism of the biosynthesis of selenocysteine in general and the mechanism of SepSecS in particular, stable tRNA super(Sec) substrates carrying aminoacyl moieties that mimic particular reaction intermediates are needed. Here, we report on the accurate synthesis of methylated, phosphorylated, and phosphonated serinyl-derived tRNA super(Sec) mimics that contain a hydrolysis-resistant ribose 3'-amide linkage instead of the natural ester bond. The procedures introduced allow for efficient site-specific methylation and/or phosphorylation directly on the solid support utilized in the automated RNA synthesis. For the preparation of (S)-2-amino-4-phosphonobutyric acid-oligoribonucleotide conjugates, a separate solid support was generated. Furthermore, we developed a three-strand enzymatic ligation protocol to obtain the corresponding full-length tRNA super(Sec) derivatives. Finally, we developed an electrophoretic mobility shift assay (EMSA) for rapid, qualitative characterization of the SepSecS-tRNA interactions. The novel tRNA super(Sec) mimics are promising candidates for further elucidation of the biosynthesis of selenocysteine by X-ray crystallography and other biochemical approaches, and could be attractive for similar studies on other tRNA-dependent enzymes. The 21st amino acid: The biosynthesis of selenocysteine (Sec) involves the enzyme O-phosphoseryl-tRNA: selenocysteinyl-tRNA synthase (SepSecS). To explore the enzyme mechanism, tRNA super(Sec) substrates carrying aminoacyl moieties that mimic particular reaction intermediates are needed. Here, we report on the solid-phase synthesis of methylated, phosphorylated, and phosphonated serinyl-derived RNA conjugates together with the corresponding tRNA super(Sec) mi