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Title:Catalysis by DNA enzymes: roles of lanthanide(III) ions and DNA aptamer modules
Author(s):Dokukin, Victor
Director of Research:Silverman, Scott K.
Doctoral Committee Chair(s):Silverman, Scott K.
Doctoral Committee Member(s):Girolami, Gregory S.; Mitchell, Douglas A.; van der Donk, Wilfred A.
Department / Program:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):DNA catalysis
DNA catalysts
DNA enzyme
Lanthanide ions
DNA aptamers
Modular catalysts
modular DNA catalysts
Abstract:For many years scientists considered DNA and RNA to be merely the means for the storage and transmission of genetic information. Catalytic roles of some RNA molecules, called ribozymes, were discovered in the early 1980s. Considering structural similarities between RNA and DNA, it was reasonable to evaluate catalytic competence of DNA. Indeed, the first deoxyribozymes were reported in 1994. However, DNA enzymes are not found in nature and can only be identified from a pool of random sequences via an artificial selection process, referred to as the in vitro selection. DNA catalysts capable of supporting various reactivities have been found. All deoxyribozymes can be classified as metalloenzymes because they require metal ion cofactors, usually at millimolar concentrations. For example, DNA catalysts for single-stranded DNA hydrolysis, previously identified in the Silverman laboratory, are functional only in presence of Mn2+ and Zn2+ cofactors at millimolar concentrations. Chapter 2 describes the discovery via the selection process of two DNA-hydrolyzing DNA enzymes that require low micromolar concentrations of a lanthanide ion as the sole polyvalent metal ion cofactor. The same selection effort also lead to the identification of a number of DNA-hydrolyzing deoxyribozymes that strictly require low micromolar lanthanide as well as millimolar Zn2+ concentrations. These DNA catalysts have a range of lanthanide dependences, including some deoxyribozymes that strongly favor one particular lanthanide ion and others that function well with more than one lanthanide ion. In addition to its catalytic competency, DNA is capable of forming strong non-Watson-Crick binding interactions with various chemical targets, including small molecules. DNA sequences possessing this ability are referred to as DNA aptamers and can also be identified via the in vitro selection process. Chapter 3 describes an effort to find DNA aptamers of ATP with a further goal of integrating these sequences into random DNA pools for deoxyribozyme selection. Providing a fixed binding module adjacent to the random region during selection was hypothesized to lead to the emergence of DNA sequences tasked exclusively with catalysis. Previously published and well-studied DNA aptamers of ATP were chosen for this task due to a lack of success in identifying new ATP binders. Chapter 4 describes selection efforts with an integrated DNA aptamer that lead to the identification of a truly modular deoxyribozyme. This DNA enzyme catalyzes tyrosine phosphorylation via the transfer of a phosphoryl group from ATP, which must be present in solution at concentrations similar to the Kd value of the integrated aptamer. Functional contribution of the binding domain to catalysis was confirmed by mutational analysis of the aptamer sequence as well as by substitution of ATP target molecules with their analogs.
Issue Date:2014-09-16
Rights Information:Copyright 2014 Victor Dokukin
Date Available in IDEALS:2014-09-16
Date Deposited:2014-08

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