Project summary/Abstract
The overall goal is to design and select two classes of metalloenzymes, metalloprotein enzymes and metallo-
DNAzymes, and to explore their applications in biocatalysis, bioimaging, and genetic engineering.
In the first project, we plan to achieve a holistic understanding of complex heteronuclear metalloenzymes
involved in multi-electron processes, specifically structural features in nitric oxide reductases (NOR), heme-
copper oxidases (HCO) and sulfite reductases (SiR) responsible for efficient and selective 2-, 4-, and 6 electron
catalytic reduction of NO, O2, and SO32-, respectively. Even though much progress has been made in studying
individual enzymes, a major gap in our knowledge is what structural features are responsible for the differences
in their functions. To fill this gap, we plan to use small and stable proteins as “scaffolds” to make “biosynthetic
models” of native enzymes with similarly high activity. By placing different heme–nonheme metal ions into the
same protein scaffold, we plan to a) understand how a heme-Cu center can exhibit either HCO or SiR activity;
b) elucidate structural features responsible for catalytic activity and substrate binding affinity in SiR; c) clarify the
roles of tyrosine in HCO and SiR activities; and d) investigate roles of heme cofactors in HCO, NOR, and SiR
activities. Accomplishing this goal will offer deeper insight into metalloprotein structure, function, and design, and
have a broad impact on biocatalysis, allowing design of biocatalysts for biochemical and biomedical applications.
In the second project, we plan to select DNAzymes with high selectivity for different metal ions with oxidation
state specificity and explore applications of these DNAzymes as imaging agents for paramagnetic metal ions
(PMIs) such as Fe and its Fe2+/Fe3+ redox cycle in living organisms. While progress has been made in developing
sensors for metal ions, sensors that can selectively detect PMIs are limited; few, if any, can detect two oxidation
states of the same metal ions simultaneously. To overcome this barrier, we have obtained DNAzymes sensors
with high selectivity for either Fe2+ or Fe3+ using in vitro selection and demonstrated imaging of both Fe2+ and
Fe3+ simultaneously in living cells using catalytic beacons. We plan to develop methods for spatiotemporal control
of DNAzyme-based imaging and for intracellular generation of DNAzymes to explore their imaging applications.
Accomplishing this goal will offer deeper insight into the roles of PMIs and their redox cycles in processes such
as ferroptosis that has been associated with neurodegenerative diseases and bacterial infections.
Finally, in a high-risk and high-return endeavor, we propose to expand DNAzyme’s applications as new
genetic engineering tools for cleaving double-stranded DNA (dsDNA) and for genome editing, as alternatives to
protein restriction enzymes and CRISPR/Cas, respectively. To achieve the goal, we plan to develop novel
peptide nucleic acid-assisted DNAzymes for dsDNA cleavage and then establish an intracellular gene-editing
platform. Achieving this goal will allow smaller and more robust DNAzymes for highly customizable recombinant
DNA cloning and high-fidelity genome editing.
