University of Illinois Urbana-Champaign

The in vitro selection and biochemical characterization of metalloDNAzymes

Ihms, Hannah

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Ihms_Hannah.pdf

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https://hdl.handle.net/2142/31993

Description

Title
The in vitro selection and biochemical characterization of metalloDNAzymes
Author(s)
Ihms, Hannah
Issue Date
2012-06-27T21:23:32Z
Director of Research (if dissertation) or Advisor (if thesis)
Lu, Yi
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
M.S.
Degree Level
Thesis
Date of Ingest
2012-06-27T21:23:32Z
Keyword(s)
  • metalloenzymes
  • in vitro selection
  • catalytic DNA
  • DNAzymes
  • metalloDNAzymes
Abstract
DNAzymes are strands of catalytic DNA. First discovered in 1994, they have proved themselves capable of catalyzing many different types of reactions with significant rate enhancements. Because they often require divalent metal-ion cofactors, DNAzymes have readily been developed into metal-ion sensors, in some cases with part-per-trillion sensitivity. These enzymes are currently isolated through in vitro selection. With little to base a DNAzyme selection’s sequence upon, in vitro selections typically begin with randomized DNA pools. As more is learned about the properties of DNAzymes, more efficient means of isolation involving rational design will become more feasible. Fundamental inquiries into the properties of heavy-metal-ion-dependent DNAzymes was the theme of this work. Heavy metal ions have significant health impacts, and thus are an active area of research in bioinorganic chemistry. Additionally, DNAzymes have proven their ability to distinguish between various metal ions with as high as million-fold selectivities. Such selectivities between metal ions with similar charge, ionic radii, and other properties are fundamentally intriguing. Co2+ and Zn2+ are two closely related metal ions, and the factors governing one DNAzyme family’s ability to distinguish between them were examined. During the course of a DNAzyme selection, it is customary to truncate the selected sequence to transform a cis-cleaving construct into a trans-cleaving construct. This general method was found to be ineffective in the case of this family, because peripheral sequences enhanced these DNAzymes’ selectivity for Co2+ over Zn2+ and Pb2+. While DNAzymes have been successfully selected against Mg2+, Zn2+, Hg22+, Mn2+/Mg3+, and other divalent cations, Cd2+-, Fe2+-, and Fe3+-dependent DNAzymes have not yet been isolated. A DNAzyme pair selective for Fe2+ and Fe3+ is of particular interest, because of their interconversion in an biological environment and the fundamental understanding a comparison of the DNAzymes selective for each would provide about DNAzymes’ abilities to distinguish between metal ions. Finally, the Pb2+--dependent DNAzyme 17E was mutated at the G1.1 position with the guanine analogs inosine, diaminopurine, and 2-aminopurine to analyze its catalytic mechanism. 17E contains the 8-17 motif that has dominated selections carried out by multiple labs under a multiplicity of conditions. By investigating the basic properties of DNAzymes, more light can be shed on the structure-function of these molecules, and expand the library of catalytic DNA ready to be used in new applications.
Graduation Semester
2012-05
Permalink
http://hdl.handle.net/2142/31993
Copyright and License Information
Copyright 2012 Hannah Ihms

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