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Title:I. In vitro selection of aptamers for perchlorate and melamine II. Towards crystallization of DNAzymes III. A highly selective lead sensor based on a classic lead DNAzyme
Author(s):Lan, Tian
Director of Research:Lu, Yi
Doctoral Committee Chair(s):Lu, Yi
Doctoral Committee Member(s):Silverman, Scott K.; Martinis, Susan A.; Nair, Satish K.
Department / Program:Biochemistry
Discipline:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):systematic evolution of ligands by exponential enrichment (SELEX)
aptamer
DNAzyme
deoxyribozyme
biosensor
melamine
Lead II Ion (Pb2+)
GR-5 DNAzyme
8-17 DNAzyme
crystallization
Abstract:Nature has given nucleic acids irreplaceable roles for all living creatures. It plays important roles in genetic information storage and transfer, as well as regulating various aspects of biology. The discovery of in vitro evolved functional nucleic acids opens a new field for nucleic acid chemistry and applications. The first in vitro isolated functional nucleic acid was discovered in 1990 through a process known as SELEX, systematic evolution of ligands by exponential enrichment. These isolated nucleic acids molecules are termed aptamer, which from the Latin word “aptus” – fit, and they possess ligand binding activity. In 1994, another class of functional nucleic acids, catalytic DNA (DNAzyme) has been isolated using a similar in vitro technique (in vitro selection) to catalyze the cleavage of a phosphodiester bond. Similar in principle, both SELEX and in vitro selection are combinatorial chemistry technique that enables the isolation of nucleic acid sequences with particular functions from an astronomical number of random sequences via iterative cycles of selection, separation and amplification. After the initial discovery of aptamers and DNAzymes, many more functional nucleic acids have been isolated for binding a wide variety of ligands and catalyzing a number of chemical transformations. The unique properties of nucleic acids have made functional nucleic acids suitable for wide range of applications, such as biosensing, drug targeting and therapeutics. Functional DNAs, due to their high stability compared to RNAs, are favored in many in vitro applications. Aptamers have been used widely in sensing applications. In theory, aptamer can be isolated for any ligands. However, in practice, aptamers for small and negatively charged anion are rarely isolated. To demonstrate possibility of isolating aptamer for such ligands, the first project aims to isolate an aptamer for a perchlorate anion (ClO4-). In addition, perchlorate is also a ubiquitous environmental contaminant. Current detection methods for perchlorate are cumbersome and not suitable for routine environmental monitoring. Isolating an aptamer for perchlorate can facilitate the development of aptamer based biosensor for more convenient environmental monitoring of perchlorate. A SELEX experiment has been carried out for isolating aptamers for perchlorate. Although a potential binder for perchlorate has been obtained, its affinity appeared to be weak. Future modifications or re-selection will be needed to improve the affinity of the aptamer. A SELEX based on the same experiment design has been carried out to isolate aptamers for melamine, which has become a threat in food safety in the U.S. and China. Two aptamers have been isolated and characterized with high affinity and selectivity. A biosensor based on personal glucose is under development using one of the aptamers, R29C33, for convenient detection of melamine. Ribozyme has been discovered for decades. However, due to the lack of 2ꞌ-OH group, DNA has once considered incapable of catalyzing any chemical reactions. With the isolation of DNAzyme, researchers have demonstrated that DNA possesses the same catalytic power that ribozyme and protein enzyme have. Although, DNAzymes have been isolated for catalyzing various types of reaction, its catalytic mechanism remains a mystery. Currently, one piece of the important information to solve the mystery is still missing, which is the three-dimensional structure for a DNAzyme in the active conformation. An immense effort has been spent on obtaining diffraction quality crystals for DNAzymes. Among all the DNAzymes attempted, including the 17E DNAzyme (an 8-17 variant), 39E DNAzyme and the GR-5 DNAzymes, only crystals for the 17E DNAzyme have been obtained. The best crystal diffracted to 7 Å but the quality of the crystal was not suitable for data collection. Future direction will be focused on getting better quality crystals for data collection. One of goals for isolating and characterizing functional DNAs is to make them more suitable for various applications. The 17E DNAzyme has been demonstrated in numerous literatures for the detection of Pb2+ due to the higher selectivity for the metal ion. However, in real world application, the 17E DNAzyme based sensor still suffers from interference due to its cross reactivity with other common metal ions at high concentrations. Interestingly, the first DNAzyme selected decades ago was using Pb2+ as the metal cofactor (we named the DNAzyme GR-5, since it was not named from the original study). Initial biochemical studies have demonstrated excellent selectivity when compared to the 17E DNAzyme. In this case, no other metal ions tests have comparable activity with Pb2+, even at much higher concentration (e.g., millimolar concentration). Based on the GR-5 DNAzyme, a much more selective and slightly more sensitive sensor for Pb2+ has been developed.
Issue Date:2013-02-03
URI:http://hdl.handle.net/2142/42441
Rights Information:Copyright 2012 Tian Lan
Date Available in IDEALS:2013-02-03
2015-02-03
Date Deposited:2012-12


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