DNAzymes as intracellular sensors for metal ion imaging and their structural characterization

Wu, Peiwen

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https://hdl.handle.net/2142/89224 Copy

Description

Title

DNAzymes as intracellular sensors for metal ion imaging and their structural characterization

Author(s)

Wu, Peiwen

Issue Date

2015-12-02

Director of Research (if dissertation) or Advisor (if thesis)

Lu, Yi

Doctoral Committee Chair(s)

Lu, Yi

Committee Member(s)

• Huang, Raven H.
• Martinis, Susan
• Smith, Andrew

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• deoxyribozymes (DNAzyme)
• Intracellular metal ion sensors

Abstract

Metal ions are essential for numerous biological processes and their regulation is crucial for maintaining normal functions. To gain a better fundamental understanding of how metal ions are regulated and where the potential molecular targets are for toxic metal ions, tools that can monitor localization and concentration of metal ions in living cells are required. Toward this goal, tremendous effort has been applied towards the development of intracellular metal ion sensors. Among them, both small molecular sensors and genetically encoded protein sensors have enjoyed the most success in intracellular metal ion sensing. A large number of sensors have been successfully used to detect metal ions that have important biological functions, such as calcium, zinc, copper and iron. At the same time, there is also emerging development in intracellular sensors for toxic metal ions, such as mercury, cadmium and lead. Despite the advances made over the previous years, it remains a significant challenge to rationally design sensors for metal ions of interest with both high sensitivity and selectivity. To meet this challenge and design sensors for a much broader range of metal ions, we and others have taken advantage of an emerging field of metalloenzymes called deoxyribozymes (DNAzymes), i.e., DNA molecules with enzymatic activities. Unlike small molecule or protein-based sensors, DNAzymes with high specificity for a specific metal ion of interest can be obtained from a combinatorial process, starting from a large DNA library containing up to 1015 different sequences. Because of such high metal ion selectivity, these DNAzymes have been converted into sensors for many metal ions, such as Pb2+, UO22+, Hg2+ and Cu2+, based on fluorescence, colorimetry, or electrochemistry. The development of these sensors has significantly expanded the range of metal ions that can be detected. The biggest advantages of this type of sensor are that it does not require advanced knowledge in order to construct a metal-binding site, and the binding affinity and selectivity toward metal ions can be fine-tuned by introducing different levels of stringency during the selection process. Moreover, it is relatively simple to synthesize DNA and many different modifications and functional groups can be easily introduced into the DNA during synthesis. Furthermore, DNA is naturally water soluble and biocompatible. All of these properties make DNAzyme sensors an attractive candidate for intracellular sensing of metal ions. However, even though DNAzymes have first been demonstrated as metal ion sensors over 10 years ago and many sensors have been reported since then, all of these sensors are limited to detecting metal ions in extracellular environments. In this dissertation, I present 1) the design, synthesis, and application of a DNAzyme-gold nanoparticle probe for uranyl detection in living HeLa cells; 2) Na+ imaging in living cells using a photocaged Na+-specific DNAzyme; and 3) fluorescent iron sensors based on Fe(II) and Fe(III) DNAzymes for iron detection in mammalian and bacterial cells. These studies demonstrated that DNAzymes, a new type of intracellular sensors, could be used as a general platform for imaging a wide range of metal ions in living organisms. Despite numerous practical applications, the mechanism of metallo-DNAzymes’ reaction and the role of metal ion in their structure and function are not yet fully understood. It remains unclear how DNA can carry out catalysis with simpler building blocks, fewer functional groups and less diverse structures than ribozymes and proteins. Similarly, the spatial arrangement of the DNAzyme enabling its superior selectivity for one metal ion over others also remains a mystery. To address these questions, an atomic resolution structure of a DNAzyme is highly desired and would greatly improve our understanding about the role of metal ion and nucleotide bases in the catalysis. However, unlike the mature fields of ribozyme and protein crystallization, DNA crystallization, especially of molecules with non-canonical structures, remains very challenging. In the last part of this dissertation, effort towards obtaining the first crystal structure of a DNAzyme in its active form is described and future directions are discussed.

Graduation Semester

2015-12

Type of Resource

text

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http://hdl.handle.net/2142/89224

Copyright and License Information

Copyright 2015 Peiwen Wu

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