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

Abstract

Direct measurement of DNArepair is critical for the annotation of their clinical relevance and the discovery of drugs for cancer therapy. Here we reported a" repaired and activated" DNAzyme (RADzyme) by incorporating as ingle methyl lesion (O 6 MeG,3 MeC,o r1 MeA) at designated positions through systematic screening.W ef ound that the catalytic activity of the RADzyme was remarkably suppressed and could be restored via enzyme-mediated DNAr epair. Benefit from these findings,af luorogenic RADzyme sensor was developed for the monitoring of MGMT-mediated repair of O 6 MeG lesion. Importantly,t he sensor allowed the evaluation of MGMT repair activity in different cells and under drugs treatment. Furthermore,a nother RADzyme sensor was engineered for the monitoring of ALKBH2-mediated repair of 3MeC lesion. This strategy provides asimple and versatile tool for the study of the basic biology of DNAr epair,c linical diagnosis and therapeutic assessment.

A simple and rapid mix-and-read assay for sensitive detection of O6-methylguanine DNA methyltransferase

“…The sensitivity of this method is 6 orders of magnitude higher than those of fluorescent methods based on Scheme 1 Principle of the mix-and-read assay for the sensitive detection of MGMT. fluorescence-switchable chemical probes (5 nM) 22 and DNAzymes (2 nM), 14 and 4 orders of magnitude higher than those of fluorescent methods based on graphene oxide (0.15 ng mL À1 ) 23 and hairpin probes (0.5 ng mL À1 ). 24 The high sensitivity of the proposed method can be ascribed to: (1) the efficient transduction of the MGMT enzyme signal into a nucleic acid signal via the PvuII-mediated cleavage of the MGMT reaction product; (2) the low background noise resulting from the good resistance of the dumbbell probe against nonspecific digestion; and (3) the high signal amplification induced by the Exo III-mediated cyclical digestion of the signal probes.…”

Recent Advances on DNAzyme‐Based Sensing

“…They obtained an activation/deactivation ratio up to 12‐fold, which was much higher than that of the previous reported H−DNAzyme [47] . Recently, our group developed a novel methyl nucleotide substituted DNAzyme which could be activated through enzyme‐mediated DNA repair process [48] . Three methylated DNA, O 6 MeG, 1MeA, and 3MeC have been systematically studied in 8–17 DNAzyme.…”

“…Hence, the precise and accurate determination of proteins or enzyme activities is important for the fundamental research and clinical diagnosis. By coupling with other recognition domains ( e. g ., aptamer and chemical reactive group), DNAzymes have been widely reported for the detection of proteins or enzyme activities in vitro and in live cells, including PAS, [63] thrombin, [64] telomerase, [65] Uracil DNA glycosylase (UDG), [66] and DNA repair enzymes [48] …”

“…Subsequently, this demethylase‐driven DNAzyme sensor was further adapted for metal ions imaging in living cells [67] . At the same time, our group presented a “repaired and activated” DNAzyme (RADzyme) by introducing a single methyl lesion through systematic screening for the monitoring of DNA repair enzymes (Figure 5B) [48] . We reasoned that a single methyl lesion, including O 6 MeG, 3MeC, and 1MeA at the selected position had a great impact on the cleavage activity, and enzyme‐mediated DNA alkylation repair could restore the catalytic activity of DNAzymes.…”

“…(B) Single methyl lesion modified fluorogenic DNAzyme sensors for DNA repair monitoring. Adapted with permission [48] . Copyright 2021, Wiley‐VCH GmbH.…”