# Spectroscopic and Mechanistic Characterization of Novel DNAzymes Selective for Redox-active Metal Ions > **NIH NIH F32** · UNIVERSITY OF TEXAS AT AUSTIN · 2024 · $78,287 ## Abstract PROJECT SUMMARY/ABSTRACT DNAzymes represent one of the most recent classes of diverse, catalytically active biomolecules. However, despite their discovery >25 years ago and exceptional potential for broad analytical and therapeutic applications, our understanding of metallo-DNAzymes in terms of binding selectivity, structure, and catalytic mechanism still lags far behind that of metalloproteins. Although DNAzymes have already been developed as highly selective metal ion sensors, the lack of fundamental knowledge regarding metallo-DNAzyme function has precluded the application of rational design to enhance metal binding affinity and specificity. The goal of this project is to obtain unparalleled insight into the structure-function relationships of metal-binding DNAzymes specific for redox-active metal ions (RAMIs) with high physiological relevance (e.g., Fe2+, Fe2+, Cu+, and Cu+2), and in turn provide a foundation for the future rational design of DNAzymes. To achieve this goal, an array of biochemical and advanced biophysical characterization techniques will be employed and cross-correlated to determine the locations of metal binding, coordination environments, binding affinities and specificities, and reaction mechanisms for a series of Fe2+, Fe3+, Cu+, and Cu+2-specific DNAzymes. Metal-bound DNAzyme resting states will be generated using a series of “non-cleavable” substrates, which will prove fundamental in determining metal binding affinities, specificities, and key spectroscopic signatures using UV-Vis/nIR, EPR, and 57Fe Mössbauer spectroscopies. By additionally applying XAS, the rudimentary coordination environment and electronic structure will be determined. Single point mutations will be screened across suspected metal-binding regions of oligonucleotide sequences, and the corresponding cleavage efficiency and characteristic spectroscopic signatures will be tracked to narrow the assignment of metal-binding site. Further advanced characterization using vibrational and pulse EPR spectroscopies will be used together with selectively isotope-labeled residues to provide a precise assignment of metal binding location and coordination environment. All of this information will be matched by computational modeling of first coordination binding models using a DFT and ab initio approaches. Lastly, a high-risk/high-reward foray will be made to grow diffraction-quality crystals for holistic structural characterization. Beyond the resting state, the mechanism of DNA/RNA cleavage by these DNAzymes will be analyzed by a combined analysis of cleaved fragment ends and careful kinetic characterization. Where necessary, rapid quench flow and rapid freeze quench methods will be employed to trap and assess potential reaction intermediates. Achieving the above goals will greatly deepen our understanding of the structure and function of metal- binding sites in DNAzymes, shifting the paradigm of metalloprotein characterization methodology to include metallo-DNA... ## Key facts - **NIH application ID:** 10897099 - **Project number:** 5F32GM145134-03 - **Recipient organization:** UNIVERSITY OF TEXAS AT AUSTIN - **Principal Investigator:** Casey Michael Van Stappen - **Activity code:** F32 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2024 - **Award amount:** $78,287 - **Award type:** 5 - **Project period:** 2022-09-05 → 2025-08-01 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/10897099 ## Citation > US National Institutes of Health, RePORTER application 10897099, Spectroscopic and Mechanistic Characterization of Novel DNAzymes Selective for Redox-active Metal Ions (5F32GM145134-03). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10897099. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*