Small Oligonucleotide Models of the Twister Ribozyme Active Site

Open Access
Author:
Tracey, Matthew Allan
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Philip C. Bevilacqua, Thesis Supervisor
  • Teh-hui Kao, Honors Advisor
Keywords:
  • RNA
  • Ribozyme
  • Twister
  • Chemistry
  • Biochemistry
  • Twister Ribozyme
  • RNA Catalysis
Abstract:
Since the initial discovery that ribonucleic acids possess catalytic activity, various examples of catalytic RNAs, or ribozymes, have been discovered. Of particular interest are those that possess self-cleaving properties such as the recently discovered twister ribozyme. While the catalytic mechanism has been thoroughly studied, much is still unknown about the biological role partly due to the fact that twister is found in all domains of life. The explicit catalytic mechanism for twister is also still unclear due to uncertainty regarding the role of a highly conserved and important adenine residue, A1. Twister is hypothesized to conduct substrate-assisted catalysis via its A1 residue, which is hypothesized to act via general acid catalysis at biological pHs assisted by small molecule buffers. As the A1 residue is covalently linked to the scissile phosphate within the active site, this makes twister unique among ribozymes as this residue may not require a complex 3D structure for optimal positioning. Therefore, I hypothesized that part of the twister catalytic mechanism could be studied in isolation via short oligoribonucleotides. The small RNA hexamers used in my studies were chosen or designed around those detailed in a previous 1992 paper whereby RNA hexamers engaged in specific self-cleavage. It was observed that hexamers with a UA/CA cleavage site were the most active and thus original oligos were designed with this in mind. This observation along with their similarity to the cleavage site of the twister ribozyme, predominately a UA or CA, made these hexamers viable models. Through my experiments, I test the viability of these models in the context of the original studies as well as in conditions and additional constructs relevant to the twister ribozyme. I find, surprisingly, that these UA and CA linkages in small hexamers do not possess intrinsic reactivity as originally determined, illustrating the importance of twister’s 3D structure to properly position the substrate for its self-cleavage mechanism.