R35GM141931

Design and Selection of Novel Metalloenzymes for Biocatalysis, Bioimaging, and Genetic Engineering - 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 DNAzyme 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 this 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.

University Of Texas At Austin

NOT APPLICABLE

93.859 - Biomedical Research and Research Training

National Institute of General Medical Sciences (NIGMS) [HHS - NIH]

Austin, Texas 787121507 United States

Single Zip Code

Maximizing Investigators' Research Award (R35 - Clinical Trial Optional) (PAR-22-180)

Amendment Since initial award the End Date has been extended from 05/31/26 to 01/31/31 and the total obligations have increased 827% from $372,117 to $3,450,251.