Heme-utilizing ribozymes and DNAzymes: Biological impacts, structural aspects, and a kinetic model of activation

Resource type
Thesis type
(Thesis) Ph.D.
Date created
2017-08-17
Authors/Contributors
Abstract
Guanine-rich RNAs and DNAs that fold into guanine quadruplexes are found to complex tightly with porphyrins such as hemin [Fe(III)-heme]. The generated complex displays robust peroxidase (1 e- oxidation) as well as peroxygenase (2 e- oxidation) activity, greater than that of disaggregated heme itself. They can, thus, be regarded as heme-Utilizing DNAzymes and ribozymes. The folded DNAzymes appear to provide a unique chemical environment to the bound heme that by analogy resembles that of hemoproteins such as horseradish peroxidase (HRP) and cytochrome P450s. This work focuses on three aspects of these ribozymes and DNAzymes. First, we demonstrate that “toxic”, guanine-rich RNAs that accumulate in the cytoplasm of neurons afflicted with the familial forms of two neurodegenerative diseases: Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD), and are indeed thought to be causative of those diseases, efficiently bind and activate heme. Second, we systematically investigate the special status (or not) of guanine quartets in DNA/RNA for the purpose of binding and activating heme. Specifically, we explore whether isoguanine-containing DNAs, which in the presence of certain cations (including Na+, Cs+ and NH4+) form isoG quintets, while in K+, they form isoG quartets, can also bind and activate heme. We make the important observation that while G-quartets and iG-quintets both bind and activate heme, iG-quartets do not. Evidence from the theoretical/computational literature provides a satisfactory explanation for this observation, which in turn helps to illuminate the key structural features of nucleic acids that are necessary for binding and activating heme. Finally, we carry out fast kinetic measurements (using a stopped-flow enabled UV-vis spectrophotometer) to study the identities and formation of hydrogen peroxide-generated activated heme species within the above DNA-heme complexes. With the aid of Pro-KIV software, we perform singular value decomposition and global fitting analysis to formulate with a kinetic scheme for heme activation by these DNAzymes.
Document
Identifier
etd10416
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Scholarly level
Supervisor or Senior Supervisor
Thesis advisor: Sen, Dipankar
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