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In vitro selection of functional DNA for molecular catalysis, conjugation, and sensing

Resource type
Thesis type
(Thesis) Ph.D.
Date created
2021-06-16
Authors/Contributors
Author: Liu, Kun
Abstract
As the central theme of this thesis research, in vitro selection has been performed to isolate functional DNAzymes and DNA aptamers for molecular catalysis and sensing. Not only details of the in vitro selection and characterization of functional DNAzymes and aptamers, but also their applications for the catalytic conjugation of biological macromolecules, immobilization of redox functionalities on surfaces, and ultrasensitive detection of total copper in industrial samples are presented. Copper is known to bind DNA on both negatively charged phosphate backbone and nucleobases; through in vitro selection, it has been demonstrated that these interactions can be coordinated in a DNAzyme to catalyze alkyne-azide cycloaddition reaction with ultralow concentrations of either Cu(I) or Cu(II) as a cofactor. The selected DNAzyme, namely, CLICK-17, catalyzes the reaction with high efficiency under both in cis (single turn-over) and in trans (multi turn-over) conditions. After characterization and kinetics studies, three applications of CLICK-17 have been explored: (1) As a biocompatible coupling reagent for labelling azide-functionalized biological macromolecules; (2) As a highly efficient co-catalyst for the immobilization of electrochemical reporters onto azide-terminated self-assembled monolayers (SAMs); (3) As a superior transducer for the construction of an electrochemical biosensor for copper. The isolation of DNA aptamers binding to small molecules is challenging, due to their lack of chemical functionalities to interact with DNA strands. Ferrocene is a particularly refractory target, as a mater of fact. Conventional in vitro selection method (ferrocene immobilized on a supporting bead) and capture selection method (DNA library immobilized on a supporting bead) in isolating aptamers binding to a water-soluble ferrocene derivative have been compared. A ferrocene binding aptamer with moderate binding affinity was isolated with capture selection method. In summary, this research achieved the in vitro selection of a DNAzyme that is useful for developing different types of applications, and a DNA aptamer that can potentially deepen our understanding of molecular recognition between a DNA aptamer and its target.
Document
Extent
163 pages.
Identifier
etd21530
Copyright statement
Copyright is held by the author(s).
Permissions
This thesis may be printed or downloaded for non-commercial research and scholarly purposes.
Supervisor or Senior Supervisor
Thesis advisor: , Hua-Zhong
Language
English
Member of collection
Download file Size
etd21530.pdf 8.79 MB

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