DNAzymes, or deoxyribozymes are DNA molecules with enzymatic activities. Since its discovery in 1994, DNAzymes have been shown to be metalloenzymes and can be converted into metal ion sensors. Scientifically, whereas a great deal of knowledge has been accumulated in the roles of metal ions in proteins, much less is known in nucleic acids. Technologically, while enormous progress has been made in the designing sensors for diamagnetic metal ions, designing sensors for paramagnetic metal ions, particularly different oxidation states of the same metal ions remains challenging. The project seeks to fill both gaps by advancing scientific knowledge of metal-binding sites in DNAzymes, and expanding their technological applications as paramagnetic metal ion sensors that will be used to improve environmental health. Specifically we first plan to employ in vitro selection to obtain DNAzymes with high activity toward phosphodiester transfer and with strong affinity for different paramagnetic metal ions (Co2+, Cu2+ or Fe2+), or different oxidation states of the same metal ion (Fe2+ vs. Fe3+). Biochemical studies of the selected DNAzymes will provide information about conserved sequence, catalytic parameters, and pH and metal ion dependence of the enzyme activity. Biophysical characterization using UV-vis, EPR, MCD, XAS, FRET, and X-ray crystallography will elucidate affinity, stoichiometry, geometry,and ligand donor sets of the metal-binding sites in these DNAzymes, as well as reaction intermediates and mechanism. The knowledge acquired in this process will be used to convert these DNAzymes into sensitive and selective metal sensors using a patented catalytic beacon technology. If the aims of this project are achieved, we will advance scientific knowledge of the roles of metal ions in each DNAzyme investigated and how different structural features influence the enzyme activity. It will bring our level of understanding of metal ions in DNAzymes closer to that in proteins. It will also allow a unique opportunity to compare and contrast structural and functional properties of the same metal ions, such as Cu2+ or Fe2+, in proteins and in DNA, which will be fascinating because proteins and DNAzymes use very different building blocks. Furthermore, the demonstration of general applicability of the patented catalytic beacon method to sense a wide variety of paramagnetic metal ions (including different oxidation states of the same metal ions) will drive the field of environmental health, allowing on-site, real-time detection of metal ions in environmental monitoring, developmental biology, clinical toxicology, wastewater treatment, and industrial monitoring. Finally, the insight gained from the study on the basic coordination chemistry will shed light on rational design of other types of metal sensors based on organic molecules, polymers or peptides. It will also have important impact on research areas beyond sensor design, such as the design of transition metal-based nucleases and pharmaceutical agents.

Public Health Relevance

Paramagnetic metal ions such as cobalt, copper and iron are beneficial to human health when low in concentration, but are toxic when high in concentration. Developing portable fluorescent DNAzyme sensors for these metal ions will advance the field of environmental health, allowing on-site, real-time detection of metal ions in environmental monitoring, developmental biology, clinical toxicology, wastewater treatment, and industrial monitoring. Insights gained from the study will shed light on rational design of other types of metal sensors and could impact on other research areas such as the design of transition metal-based nucleases and pharmaceutical agents.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES016865-05
Application #
8272648
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Balshaw, David M
Project Start
2008-07-01
Project End
2014-04-30
Budget Start
2012-05-01
Budget End
2014-04-30
Support Year
5
Fiscal Year
2012
Total Cost
$350,082
Indirect Cost
$110,859
Name
University of Illinois Urbana-Champaign
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
041544081
City
Champaign
State
IL
Country
United States
Zip Code
61820
Wu, Peiwen; Yu, Yang; McGhee, Claire E et al. (2014) Applications of synchrotron-based spectroscopic techniques in studying nucleic acids and nucleic acid-functionalized nanomaterials. Adv Mater 26:7849-72
Xiang, Yu; Wu, Peiwen; Tan, Li Huey et al. (2014) DNAzyme-functionalized gold nanoparticles for biosensing. Adv Biochem Eng Biotechnol 140:93-120
Xiang, Yu; Lu, Yi (2014) DNA as sensors and imaging agents for metal ions. Inorg Chem 53:1925-42
Hwang, Kevin; Wu, Peiwen; Kim, Taejin et al. (2014) Photocaged DNAzymes as a general method for sensing metal ions in living cells. Angew Chem Int Ed Engl 53:13798-802
Xing, Hang; Hwang, Kevin; Li, Ji et al. (2014) DNA Aptamer Technology for Personalized Medicine. Curr Opin Chem Eng 4:79-87
Torabi, Seyed-Fakhreddin; Lu, Yi (2014) Functional DNA nanomaterials for sensing and imaging in living cells. Curr Opin Biotechnol 28:88-95
Zhou, Zhaojuan; Xiang, Yu; Tong, Aijun et al. (2014) Simple and efficient method to purify DNA-protein conjugates and its sensing applications. Anal Chem 86:3869-75
Wu, Peiwen; Hwang, Kevin; Lan, Tian et al. (2013) A DNAzyme-gold nanoparticle probe for uranyl ion in living cells. J Am Chem Soc 135:5254-7
Li, Le-Le; Xie, Mengying; Wang, Jie et al. (2013) A vitamin-responsive mesoporous nanocarrier with DNA aptamer-mediated cell targeting. Chem Commun (Camb) 49:5823-5
Li, Le-Le; Wu, Peiwen; Hwang, Kevin et al. (2013) An exceptionally simple strategy for DNA-functionalized up-conversion nanoparticles as biocompatible agents for nanoassembly, DNA delivery, and imaging. J Am Chem Soc 135:2411-4

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