Syn Bases: Their Prevalence, Relevance, and Functional Utility in Functional RNA

Open Access
Author:
Reigh, Stephanie Ann
Graduate Program:
Chemistry
Degree:
Master of Science
Document Type:
Master Thesis
Date of Defense:
August 27, 2009
Committee Members:
  • Philip C. Bevilacqua, Thesis Advisor
Keywords:
  • RNA
Abstract:
Due to a high number of rotatable bonds in both the ribose sugar and phosphate backbone, nucleotides in RNA can occupy a wide ensemble of conformational states. One conformational state of interest is when a base takes the syn conformation, in which the base resides over the sugar and the Watson-Crick face of a nucleotide is positioned towards the phosphate backbone. I show herein that the syn conformation is common in functional RNA, often in functional locations in riboswitches, aptamers, and ribozymes. In the hepatitis delta virus ribozyme, as an example, only one base in 100 takes the syn conformation, but mutation of that base reduces catalytic activity as much as 3000-fold. Syn bases cluster in the binding pockets of both the lysine riboswitch and the malachite green aptamer, participating in stacking and hydrogen bonding interactions with their respective ligands. To further investigate the utility of syn bases in functional RNA, conformationally restricted nucleotides (CRNs) are used to populate the native state, either through stabilization of the native state or destabilization of a misfolded state. 8-bromopurines can be successfully incorporated into RNA during transcription, and these CRNs favor the syn conformation. These CRNs have already been incorporated systematically to improve kinetics in the leadzyme system. I present preliminary evidence that 8BrGTP and 8BrATP can be incorporated during transcription. Future directions of this project will incorporate CRNs at random sites to see whether function can be restored or enhanced from syn base insertion.