Control of Biological Processes with Modified Nucleosides, Amino Acids, and Small-Molecule Probes

Abstract

Several novel caging groups with a wide range of applications were designed. The caging groups were applied to the synthesis of molecular tools for control of gene expression. A number of RNA, DNA, and antisense-oligomer subunits were prepared. The caged nucleosides were site-specifically incorporated into corresponding oligonucleotide strands, using standard automated-synthesis protocols. The caged nucleosides were found to readily photolyse under biologically non-damaging UV-light (>365 nm). Spatio-temporal control over oligomers’ biological and/or catalytic activity was demonstrated with excellent on(or off) photo-triggered switches. Additionally, non-standard nucleosides 5-formylcytidine and N-[(9-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl]threonine were synthesized for incorporation into tRNA to elucidate the importance of the non-standard nucleoside modifications on codon recognition in RNA translation. Furthermore, tools for control over protein functions were designed. A set of novel unnatural amino acids were successfully synthesized and genetically (in vivo) incorporated into proteins using evolved orthogonal tRNA/synthetase pairs. Lastly, several caged protein kinase inhibitors were prepared, and used to study importance of kinase enzymatic activity in embryonic development of Xenopus laevis.