Files in this item
|(no description provided)|
|Title:||Deoxyribozyme-Mediated Synthesis of Branched Nucleic Acids and Application to Modulation of Ribozyme Catalysis|
|Doctoral Committee Chair(s):||Silverman, Scott K.|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||Deoxyribozymes are single-stranded DNA molecules that catalyze a variety of chemical reactions. Catalytic DNAs have not been found in nature but can be identified by in vitro selection from random sequences. In this thesis, deoxyribozymes that ligate two RNA substrates or attach DNA to RNA are identified and characterized. These studies have been useful for understanding fundamental aspects of nucleic acid catalysis and for providing practical reagents.
Ribozymes are RNA molecules that possess catalytic activity. In order to perform their functions, RNA molecules must fold into specific three-dimensional structures. RNA folding landscapes are very rugged, and RNA molecules have a strong tendency to form transient metastable conformations that represent local minima on the RNA folding landscape. In nature, these misfolded states can represent kinetic traps in which RNA molecules remain for some time before they fold correctly. One of our goals is to gain fundamental understanding of RNA folding pathways through the use of the DNA constraint strategy, in which two attached complementary DNA strands form a duplex that can be used as a structural constraint to control RNA conformation. In an initial study, we employed the DNA constraint strategy to control the catalytic activity of a small self-cleaving catalytic RNA, the hammerhead ribozyme. To prepare the DNA-modified hammerhead ribozyme, we used in vitro selection to identify the 9FQ4 deoxyribozyme that allows attachment of DNA to RNA at branch-site adenosine in high yield and with useful generality. We showed that when two complementary DNA strands were attached by 9FQ4 at two distinct positions of cis or trans hammerhead ribozyme constructs, the ribozyme catalytic activity was substantially diminished. Subsequent addition of free DNA oligonucleotide that was complementary to one of the constraint strands or addition of DNase I restored the high catalytic activity of the ribozyme. In contrast, attachment of two non-complementary strands at the same positions had only a modest effect on catalysis. Experiments using RNase T2 demonstrated that the secondary structure of the ribozyme was intact, indicating that the tertiary structure was affected by the DNA constraint.
We have also used the 9FQ4 deoxyribozyme to attach DNA strands to the large multi-domain L-21 ScaI ribozyme form of the Tetrahymena group I intron. DNA strands were placed at seven pairs of target sites. Similar to the results obtained for the hammerhead ribozyme, when the two attached DNA strands were complementary, ribozyme catalysis was nearly completely abolished. Activity was restored to the wild-type level after separation of the constraint strands by addition of free complementary DNA. Together, the data obtained for the hammerhead and group I intron ribozymes validate the DNA constraint approach to create kinetically stable discrete misfolded states of RNAs. We anticipate that further studies using these DNA-constrained RNAs will contribute to our fundamental knowledge of RNA molecular biology.
Adenosine is the conserved nucleotide at the branch site in most pre-mRNA introns, where its 2'-hydroxyl group acts as a nucleophile for the first step of splicing. The 7S11 deoxyribozyme also forms its branched RNA product by mediating the nucleophilic attack of a 2'-hydroxyl of an internal adenosine on a 5'-triphosphate. Because the branch-site adenosine used by 7S11 was not predetermined by the selection design, it is not clear whether this preference is an inherent chemical characteristic of forming branched RNA or a coincidental outcome of the particular selection experiment. To explore this, we designed a new in vitro selection experiment based on the three-helix-junction structural format of 7S11, but provided branch-site uridine during the selection process. The resulting deoxyribozymes had a substantial preference for a branch-site adenosine, although adenosine was never available during the selection itself. This suggests a chemical basis for nature's choice of the branch-site nucleotide.
Finally, we intend to study the role that divalent metal ions play in the phosphoryl transfer reaction during RNA splicing by group II introns. To enable these studies, in vitro selection was initiated to identify deoxyribozymes that mediate the nucleophilic attack of an internal free 3'-OH and also tolerate sulfur substitution of the 2'-oxygen at the branch-site adenosine. The selection efforts instead produced a collection of deoxyribozymes that utilize alternative internal nucleotides as branch sites for ligation.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2008.
|Date Available in IDEALS:||2014-12-17|