Folding‐upon‐Repair DNA Nanoswitches for Monitoring the Activity of DNA Repair Enzymes

Abstract

We present anew class of DNA-based nanoswitches that, upon enzymatic repair,c ould undergo ac onformational change mechanism leading to ac hange in fluorescent signal. Such folding-upon-repair DNAn anoswitches are synthetic DNAs equences containing O 6-methyl-guanine (O 6-MeG) nucleobases and labelled with af luorophore/quencher optical pair.T he nanoswitches are rationally designed so that only upon enzymatic demethylation of the O 6-MeG nucleobases they can form stable intramolecular Hoogsteen interactions and fold into an optically active triplex DNAs tructure.W e have first characterized the folding mechanism induced by the enzymatic repair activity through fluorescent experiments and Molecular Dynamics simulations.W et hen demonstrated that the folding-upon-repair DNAn anoswitches are suitable and specific substrates for different methyltransferase enzymes including the human homologue (hMGMT) and they allow the screening of novel potential methyltransferase inhibitors.

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

“…[7] Engineered nucleic acids have emerged as valuable arsenals for developing fluorescent probes for the measurement of DNA damage repair. [8][9][10][11][12][13] However,o nly very few nucleic acid probes have been demonstrated for live cell applications.A prominent design is the fluorescence-based multiplex flowcytometric host cell reactivation assay (FM-HCR), which uses plasmid reporters bearing DNAl esions to measure DNA repair capacity in live cells. [9] However,the genetic reporters require labor-a nd cost-intensive synthesis of plasmids with DNAl esions.A na lternative strategy is to exploit af luorogenic nucleoside analog,t he fluorescence of which is quenched by as pecific 1MeA lesion to monitor ALKBH3 repair activity.…”

Harnessing an emissive guanine surrogate to design small-molecule fluorescent chemosensors of O6-methylguanine-DNA-methyltransferase (MGMT)

“…In this design, the reaction of the probe with MGMT leads to a transfer of the O 6 -substituent from the substrate to the protein, leading to disruption of the FRET pair and an increase of fluorescence signal. 12,13 Alternatively, MGMT-promoted dealkylation of O 6 -alkylated oligonucleotide substrates can be exploited to trigger a conformation switch, leading to a change of a fluorescence signal 14,15 or activation of a DNAzyme cleaving a fluorogenic RNA substrate. 16 However, a common drawback of oligonucleotide-based probes is their poor cellular permeability, making their use in live cells problematic.…”

Reconstructing the Free Energy Profiles Describing the Switching Mechanism of a pH-Dependent DNA Nanodevice from ABMD Simulations

“…Recently, a O6-methyl-guanine 2 of 12 (O6-MeG) modified triple helix-based nanoswitch was designed, with the ability to monitor the activity of DNA repair enzymes. [25] In this context, the full understanding of the DNA triplex-based devices' atomistic behavior represents an important aspect for a fine prediction of the rules governing their structure/dynamics/function relationship. In the last few years, we demonstrated the importance of Molecular Dynamics (MD) simulations coupled to experiments in describing and predicting the atomistic behavior of DNA nanoswitches [26,27] integrated into complex nanostructures [28].…”