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Title:Cellular metal ion sensing using DNAzymes
Author(s):Hwang, Kevin
Director of Research:Lu, Yi
Doctoral Committee Chair(s):Lu, Yi
Doctoral Committee Member(s):Chan, Jefferson; Murphy, Catherine J; Suslick, Kenneth S
Department / Program:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
metal ion sensing, fluorescent sensors
bioinorganic chemistry
Abstract:Metal ions are important elements in biology and are involved in numerous reactions essential for maintaining life on earth. Most commonly, metal ions confer their role as structural elements or catalytic cofactors within metalloproteins. Metalloproteins comprise more than half of all known proteins and are at the heart of several important biological processes including photosynthesis, respiration, and nitrogen fixation. Another equally important role of metal ions is as signaling molecules. It has been shown that changes in concentrations of calcium, zinc, or copper can trigger downstream signaling effect in neurons. Despite these important functions, high levels of many metal ions, especially transition metal ions, are known to be toxic to cells. Thus, cells have adapted strategies to finely regulate the uptake, storage, and distribution of metal ions within different compartments. The importance of these mechanisms for maintaining metal ion homeostasis within cells and the key role of metal ions in life have been of major interest for years. However, the understanding of these mechanisms and how metal ions play their role is largely unknown in many cases. An important step towards advancing our understanding of metal ions is developing the ability to measure their concentrations in different cellular compartments with accuracy and sensitivity. To achieve this goal, several techniques are available that have greatly advanced our understanding of metal ions. However, current methods suffer from a number of limitations that have slowed progress. Analytical tools such as ICP-MS and AAS, while effective at measuring the cocnentrations of metal ions very sensitively, usually work in bulk and thus are not easily amenable to single cell studies let alone the distribution in different cellular compartments. Moreover, these techniques are often unable to distinguish between different oxidation states of a metal ion or between bound and mobile forms. Techniques based on X-ray absorption, such as X-ray fluorescence microscopy (XFM) can simultaneously detect multiple metal ions and can distinguish between different oxidation states. However, these techniques require the use of highly focused X-ray beams limiting more widespread availability of such techniques. Furthermore, biological samples cannot be examined in a real time manner, and the obtained distribution of metal ions represents only total metal content, without regard to whether the metals are easily exchangeable or are tightly bound, a major factor determining biological activity. To overcome these issues several metal ion sensors have been designed based on either small molecules or proteins with great sensitivities of detection. The use of such sensors has provided great insight into the functions of metal ions. However, as these sensors are usually rationally designed through a trial and error process, it is challenging to generalize the successful designs to sense other metal ions or to meet new desired criteria. DNAzymes, DNA sequences with catalytic activity, have recently emerged as an alternative class of sensors for metal ions. As selection of DNAzymes for different metal ions is carried out through a combinatorial process, new sequences with desired activity and selectivity can be easily obtained by changing the selection conditions. Many DNAzymes have been selected with high sensitivity for different metal ions including zinc, copper, lead, and uranyl. Surprisingly, despite the promise of using DNAzymes as sensors for cellular metal ions, almost all applications of DNAzyme-based sensors until now have been in environmental metal ion detection and DNAzyme-based sensors for cellular metal detection have only very recently been explored as an option. The goal of my PhD research was to establish novel strategies to make DNAzymes viable sensors for cellular metal ion detection. Chapter 1 briefly describes the background and overall aims of my research. In chapter 2, I describe my efforts in designing "caged" DNAzyme-based sensors that can be activitated by light when they reach the desired cellular compartments. This method reduces the off-target signals and as demonstrated in this chapter is highly generalizable to other DNAzyme-based sensors. In chapter 3, I explain my efforts in designing a ratiometric DNAzyme-based sensor to enable quantitative measurement within the cells. Finally, chapter 4 summarizes my efforts in enhancing the caging strategy to enable better stability, faster decaging, or the use of long-wavelength light for decaging by using lanthanide-doped metal nanoparticles.
Issue Date:2016-01-25
Rights Information:Copyright 2016 Kevin Hwang
Date Available in IDEALS:2016-07-07
Date Deposited:2016-05

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