Date of Award
Doctor of Philosophy (PhD)
Kathryn B. Grant
The atom-specific modification has been extensively applied in RNA function and structure investigations, catalysis analysis, mechanism studies, as well as therapeutics discoveries. Selenium-modified uridine (SeU-RNA) is one of the naturally occurring modifications that was discovered in bacterial tRNAs (SeU-RNA) at the wobble position of the anticodon loop. Its exact role in the RNA-RNA interaction, especially during the mRNA decoding is not completely understood but it was proposed that such Se derivatization on tRNAs probably improves the accuracy and efficiency of base-pairing. The wobble base pairs, where U in RNA (or T in DNA) pairs with G instead of A, might compromise the high specificity of the base pairing. The U/G wobble pairing is ubiquitous in RNA, especially in non-coding RNA. To assist the research exploration, we have hypothesized to discriminate against U/G wobble pair by tailoring the steric and electronic effects at the 2-exo position of uridine base and replacing 2-exo oxygen with a selenium atom. This oxygen replacement with selenium offers a unique chemical strategy to enhance the base pairing specificity at the atomic level. Here, we report the first synthesis of the 2-Se-U-RNAs through synthetic incorporation of 2-Se-uridine (SeU) phosphoramidite as well as enzymatic incorporation of 2-Se-uridine triphosphate. Our biophysical and structural studies of the SeU-RNAs indicate that this single atom replacement can indeed create a novel U/A base pair with higher specificity than the natural one. We reveal that the SeU/A pair maintains a structure virtually identical to the native U/A base pair, while discriminating against U/G wobble pair. Moreover, we have demonstrated that the synthesized SeUTPs (2-Se-UTP and 4-Se-UTP) are stable and recognizable by T7 RNA polymerase. Furthermore, the transcribed SeU-hammerhead ribozyme has the similar activity as the corresponding native, which suggests usefulness of SeU-RNAs in function and structure studies of noncoding RNAs, including the Se-tRNAs.
Sun, Huiyan, "Atom-Specific Modification of Uracil Bases with Selenium for RNA Structure and Function Studies." Dissertation, Georgia State University, 2014.