Directed Evolution of a G‑Quadruplex Peroxidase DNAzyme and Application in Proteomic DNAzyme–Aptamer Proximity Labeling

DNAzymes
have been limited in application by their low
catalytic
rates. Here, we evolved a new peroxidase DNAzyme mSBDZ-X-3 through
a directed evolution method based on the capture of self-biotinylated
DNA catalyzed by its intrinsic peroxidase activity. The mSBDX-X-3
DNAzyme has a parallel G-quadruplex structure and has more favorable
catalytic properties than all previously reported peroxidase DNAzyme
variants. We applied mSBDZ-X-3 in an aptamer-coupled proximity-based
labeling proteomic assay to determine the proteins that bind to cell
surface cancer biomarkers EpCAM and nucleolin. Confocal microscopy,
western blot analysis, and LC–MS/MS showed that the hybrid
DNAzyme aptamer-coupled proximity assay-labeled proteins associated
with EpCAM and nucleolin within 6–12 min in fixed cancer cells.
The labeled proteins were identified by mass spectrometry. This study
provides a highly efficient peroxidase DNAzyme, a methodology for
selection of such variants, and a method for its application in spatial
proteomics using entirely nucleic acid-based tooling.