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

Abstract

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.

Development of Better Aptamers: Structured Library Approaches, Selection Methods, and Chemical Modifications

“…It is notable that the G‐rich random domain alone was sufficient to generate aptamers with a defined G‐quadruplex structure. This was also the case for a recently developed G‐quadruplex peroxidase DNAzyme derived from a guanine‐rich library whose random domain contained 42 % guanine [101] . These findings demonstrate that G‐quadruplexes need not be directly pre‐engineered into the starting library.…”

“…This was also the case for a recently developed Gquadruplex peroxidase DNAzyme derived from a guaninerich library whose random domain contained 42 % guanine. [101] These findings demonstrate that G-quadruplexes need not be directly pre-engineered into the starting library. Even basic rational design strategies, such as increasing the percentage of guanine residues in the random region, can enhance the functional capacity of DNA libraries.…”

CRISPR/Cas14 and G-Quadruplex DNAzyme-Driven Biosensor for Paper-Based Colorimetric Detection of African Swine Fever Virus

“…It may be attributed to the presence of dithiothreitol, a reducing agent incorporated in the storage buffer designed to safeguard the activity of Cas14a, directly scavenging the ABTS-free radicals. 37 To further improve the naked-eye detectability of the colorimetric signal using the CRISPR/Cas14a-G4 biosensor, we optimized the concentration of G4 ranging from 50 to 600 nM. As observed in Figure 3b, the maximum visual contrast between positive and negative groups was obtained when the concentration of G4 reached 400 nM.…”

“…Excessive Cas14/sgRNA complexes notably reduced the absorbance signal. It may be attributed to the presence of dithiothreitol, a reducing agent incorporated in the storage buffer designed to safeguard the activity of Cas14a, directly scavenging the ABTS-free radicals …”

Selective Hemin Binding by a Non‐G‐quadruplex Aptamer with Higher Affinity and Better Peroxidase‐like Activity

“…[43] Second, our buffer did not contain K + , which might be another reason for the suppression of G4 sequences. Finally, some previous hemin selections used biased library already rich in guanine (Table S4), [15,44] and this also contributed to the repeated isolation of G4.…”