Orthogonal Demethylase-Activated Deoxyribozyme for Intracellular Imaging and Gene Regulation

Abstract

The epigenetic modification of nucleic acids represents a versatile approach for achieving high-efficient control over gene expression and transcription and could dramatically expand their biosensing and therapeutic applications. Demethylase-involved removal of N6-methyladenine (m 6 A) represents one of the vital epigenetic reprogramming events, yet its direct intracellular evaluation and as-guided gene regulation are extremely rare. The endonuclease-mimicking deoxyribozyme (DNAzyme) is a catalytically active DNA that enables the site-specific cleavage of the RNA substrate, and several strategies have imparted the magnificent responsiveness to DNAzyme by using chemical and light stimuli. However, the epigenetic regulation of DNAzyme has remained largely unexplored, leaving a significant gap in responsive DNA nanotechnology. Herein, we reported an epigenetically responsive DNAzyme system through the in vitro selection of an exquisite m 6 A-caged DNAzyme that could be specifically activated by FTO (fat mass and obesity-associated protein) demethylation for precise intracellular imaging-directed gene regulation. Based on a systematic investigation, the active DNAzyme configuration was potently disrupted by the site-specific incorporation of m 6 A modification and subsequently restored into the intact DNAzyme structure via the tunable FTO-specific removal of m 6 A-caging groups under a variety of conditions. This orthogonal demethylase-activated DNAzyme amplifier enables the robust and accurate monitoring of FTO and its inhibitors in live cells. Moreover, the simple demethylase-activated DNAzyme facilitates the assembly of an intelligent self-adaptive gene regulation platform for knocking down demethylase with the ultimate apoptosis of tumor cells. As a straightforward and scarless m 6 A removal strategy, the demethylase-activated DNAzyme system offers a versatile toolbox for programmable gene regulation in synthetic biology.

“Repaired and Activated” DNAzyme Enables the Monitoring of DNA Alkylation Repair in Live Cells

“…Interestingly,asingle 1MeA lesion at position A6 showed little impact on the activity of 8-17 DNAzyme,t hough ar ecent study reported that am6A modification at this position greatly inhibited the activity. [27] Thed istinction could be attributed to the high sensitivity of 8-17 DNAzyme to chemical microenvironments in the catalytic core,suggesting acrucial role of N 6 site other than N 1 site of A6 in the catalysis.T ogether, four DNAzymes (3MeC4, 3MeC9, 3MeC13, and 1MeA10) with very low catalytic activities were obtained, which conferred viable candidates for the development of RADzyme sensors for monitoring ALKBH mediated DNArepair.…”

Recent Advances on DNAzyme‐Based Sensing

“…Recently, a demethylase‐activated DNAzyme sensor has been reported by the Wang group for intracellular FTO imaging (Figure 5A) [49] . This study showed that a m 6 A‐modification in catalytic core could efficiently block the catalytic activity of DNAzymes, and the removal of the methyl group by FTO could further restore their normal function.…”

“…Three methylated DNA, O 6 MeG, 1MeA, and 3MeC have been systematically studied in 8–17 DNAzyme. The Wang lab also reported an FTO activated DNAzyme containing a m 6 A modification through solid‐pahse synthesis [49] . The catalytic activity of these DNAzymes could be remarkably suppressed with a single methylation at the selected position, and enzyme‐mediated demethylation process could restore their activities.…”

“…(A) Demethylase‐activated DNAzyme for endogenous FTO imaging and gene regulation. Adapted with permission [49] . Copyright 2021, American Chemical Society.…”

DNA-based enzymatic systems and their applications

“…In their pioneer work, DNA strands with unique secondary structures can catalyze the transesterification of RNA phosphate diester bonds. Since then, a plethora of artificial DNA sequences as active enzymes were selected by Systematic Evolution of Ligands by Exponential enrichment (SELEX), which has become an important technology for Dzs library screening due to its excellent target analysis range and strong affinity ( Silverman, 2016 ; Kumar et al., 2019 ; Wang et al., 2021a ). Regioselective Dzs consisting of synthetic DNA single stands and different cofactors have been developed with diverse catalytic abilities ( Kumar et al., 2019 ; Hollenstein, 2015 ), e.g., DNA/RNA cleavage or ligation ( Zhou et al., 2015 , 2016 ; Praetorius et al., 2017 ; Wu et al., 2017 ; Huang et al., 2020 ; Purtha et al., 2005 ), phosphorylation ( Camden et al., 2016 ), hydrolysis ( Chandra et al., 2009 ), and alkyne-azide “click’ cycloaddition ( Liu et al., 2020a ).…”