Please use this identifier to cite or link to this item:
|Title:||CHARACTERIZATION AND APPLICATION OF SELF-PHOSPHORYLATING DEOXYRIBOZYMES|
|Authors:||McManus, Simon A.|
|Keywords:||quadruplex;biosensor;DNAzyme;SELEX;Biochemistry, Biophysics, and Structural Biology;Biochemistry, Biophysics, and Structural Biology|
|Abstract:||<p>The process of in vitro selection has led to the isolation of many catalytic DNA molecules, called deoxyribozymes, which can catalyze a range of biologically-relevant reactions. Despite these advances, questions still remain as to why DNA, which seems more suited to information storage than catalysis can efficiently catalyze chemical reactions. In this thesis, a group of deoxyribozymes that can catalyze their own phosphorylation using NTP substrates are used a model system to study how DNA is able to fold into complex structures necessary for catalysis. Using a variety of structural probing techniques, these studies elucidated a common secondary structure shared by three deoxyribozymes, which do not appear to share a common ancestor sequence. This suggests that this motif may be most efficient motif to catalyze self-phosphorylation by DNA. It also more generally demonstrates that DNA can undergo convergent evolution to reach the same complex folding arrangement. A fourth deoxyribozyme was found to fold into a complex tertiary structure containing a novel quadruplex-helix pseudoknot motif. The finding of this pseudoknot and comparison with other quadruplexes found in other functional nucleic acids led us to investigate whether these stable motifs could be incorporated into nucleic acid libraries to improve the process of in vitro selection and give researchers a better chance of isolating functional nucleic acids. Design and characterization of structured libraries revealed that DNA libraries could be made in which the majority of sequences are folded into quadruplex arrangements. The incorporation of this quadruplex scaffold into DNA sequence libraries may ease the isolation of functional nucleic acids that contain this useful structural motif. In the final part of this thesis, a self-phosphorylating deoxyribozyme was converted from a tool for study of DNA structure to a sensor for GTP and Mn<sup>2+</sup>, demonstrating that deoxyribozyme substrates can be converted into targets for biosensors.</p>|
|Appears in Collections:||Open Access Dissertations and Theses|
Items in MacSphere are protected by copyright, with all rights reserved, unless otherwise indicated.