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Review
. 2018 Jul 20;13(7):1721-1733.
doi: 10.1021/acschembio.7b00919. Epub 2017 Nov 30.

Fluorescent Probes of DNA Repair

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Free PMC article
Review

Fluorescent Probes of DNA Repair

David L Wilson et al. ACS Chem Biol. .
Free PMC article

Abstract

DNA repair is now understood to play a key role in a variety of disease states, most notably cancer. Tools for studying DNA have typically relied on traditional biochemical methods which are often laborious and indirect. Efforts to study the biology and therapeutic relevance of DNA repair pathways can be limited by such methods. Recently, specific fluorescent probes have been developed to aid in the study of DNA repair. Fluorescent probes offer the advantage of being able to directly assay for DNA repair activity in a simple, mix-and-measure format. This review will summarize the distinct classes of probe designs and their potential utility in varied research and preclinical settings.

Figures

Figure 1
Figure 1
Biochemical activities of key classes of repair enzymes. The lesions being recognized are marked by red stars.
Figure 2
Figure 2
Transduction of repair activity into a fluorescence signal. The lesion containing oligonucleotide is designed such that the lesion inhibits signal generation. Repair of the lesion is the initiating step in a transduction cascade that generates a fluorescent output. In certain probe designs, lesion repair alone generates a direct fluorescent output. In most cases, however, after the lesion is repaired there are several additional steps required to transduce the repaired oligonucleotide into a fluorescent signal.
Figure 3
Figure 3
Examples of major types of DNA repair probe designs. (a) Simple molecular beacon probe. (b) DNA binding-stimulated fluorescence probe. (c) Naturally quenched probe. (d) Host cell reactivation probe. (e) O6-guanine labeled probe. (f) Graphene oxide based probe.
Figure 4
Figure 4
(a) The molecular beacon based probe reported by Saparbaev, which uses APE1 to generate a signal following the repair activity of hUNG. (b) The molecular beacon based probe reported by Lloyd uses a simple duplex and requires the AP lyase activity of OGG1 to generate a signal.
Figure 5
Figure 5
Signal amplification cycle of a nuclease or DNAzyme dependent signal amplification probe. The template strand is generated as a product of DNA repair.
Figure 6
Figure 6
Example of a signal amplification probe of TDG reported by Chen et al. Upon repair of the G:T mismatch by TDG, the AP endonuclease EnIV cuts the DNA. The resulting 5′ terminal is digested by T7Exo. The G-containing template strand duplexes with the reporter oligonucleotide that is digested by T7 Exo to generate a fluorescent signal. Because the template strand is recycled, the signal of a single enzyme turnover is amplified. Reproduced from ref 42 with permission of The Royal Society of Chemistry, copyright 2013.
Figure 7
Figure 7
T4 PNK copper nanoparticle dumbbell probe designed by Qing et al. In order to assay for the presence of T4 PNK, T4 DNA ligase and an exonuclease are added to the mixture followed by the nicked dumbbell. Only in the presence of T4 PNK will the 5′ OH be phosphorylated, allowing for ligation of the nick which protects the structure from digestion. Ascorbate and Cu2+ are then added, which template fluorescent copper nanoparticles in the presence of duplexed DNA.
Figure 8
Figure 8
Several examples of fluorescent nucleoside analogue bases. Each base is attached to deoxyribose.
Figure 9
Figure 9
Base flipping probes measure the dynamics of base flipping enzymes using environmentally sensitive fluorescent nucleoside analogues.
Figure 10
Figure 10
A damaged base quenching probe of the demethylase ABH3, which relies on the ability of 1-methyladenine (m1A) to quench the fluorescence of a neighboring pyrene nucleoside analogue. Upon demethylation by ABH3, the quenching interaction is diminished owing to the loss of a formal positive charge on the damaged base.
Figure 11
Figure 11
O6-Benzylguanine labeled probes. The red portion of each molecule is transferred to a reactive cysteine residue in the active site of MGMT. (a) The dabcyl labeled O6-benzyl deoxyguanosine reported by Beharry and co-workers. The modified nucleoside is incorporated into fluorescently labeled oligonucleotides. MGMT activity removes the quenching dabcyl moiety. (b) The rotor based probe reported by Yu and co-workers. Upon transfer of the CCVJ moiety to MGMT, intramolecular bond rotation about the trisubstituted olefin is hindered causing CCVJ to become emissive.

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