In Vitro Fluorogenic Real‐Time Assay of the Repair of Oxidative DNA Damage

Abstract

The repair of oxidative damage to DNA is essential to avoidance of mutations that lead to cancer. Oxidized DNA bases, such as 8-oxoguanine, are a chief source of these mutations, and the enzyme 8-oxoguanine glycosylase 1 (OGG1) is the chief human enzyme that excises 8-oxoguanine from DNA. The activity of OGG1 has been linked to human inflammation responses and to cancer, and researchers are beginning to search for inhibitors of the enzyme. However, measuring the activity of the enzyme typically requires laborious gel-based measurements of radiolabeled DNAs. Here we report on the design and properties of fluorogenic probes that directly report on OGG1 (and bacterial homologue Fpg) activity in real time as the oxidized base is excised. The probes are short modified DNA oligomers containing fluorescent DNA bases and are designed to utilize the damaged DNA base itself as a fluorescence quencher. Screening of combinations of fluorophores and 8-oxoguanine revealed two fluorophores, pyrene and tCo, that are strongly quenched by the damaged base. We tested 42 potential probe designs containing these fluorophores, and we found an optimized probe OGR1 that yields a 60-fold light-up signal in vitro with OGG1 and Fpg, and can report on oxidative repair activity in mammalian cell lysate and with bacterial cells overexpressing a repair enzyme. Such probes may be useful in quantifying enzyme activity and performing competitive inhibition assays.

Potent and Selective Inhibitors of 8-Oxoguanine DNA Glycosylase

“…The amount of increase is similar to or greater than that provided by a positive control of oxidatively damaging conditions. We also employed a fluorescence probe of OGG-1 activity 39 to directly test the ability of SU0268 to inhibit OGG1 activity in HeLa and MCF-7 tumor cell lysates lines, which have relatively low and high OGG1 activities, respectively. Results of the assay are shown in Figure 8b; the data confirm the inhibition of OGG1 activity at 0.5 μ M of the compound, amounting to 64% and 95% inhibition of probe signal in the two lysates as measured at 20 h. Thus, the collective data provide evidence that SU0268 ( 41 ) can be active at the intended cellular target, both in lysates and in living cells.…”

“…After that, OGR1 probe 39 (1.2 μ M) was added to the reaction mixture. Fluorescence at 460 nm was measured on a Thermo Fluoroskan Ascent FL fluorescence plate reader ( λ ex = 355 nm).…”

“…(a) Incubation with 0.5 μ M SU0268 increases the number of 8-oxodG lesions in HeLa cell DNA. (b) SU0268 (0.5 μ M) inhibits OGG1 activity as measured by fluorescence probe (pOGR1) 39 in HeLa and MCF-7 cell lysates.…”

In Situ Detection of Complex DNA Damage Using Microscopy: A Rough Road Ahead

“…Since non-covalent binding is weaker, it allows the renewal of fluorogen molecules avoiding photobleaching. This technique also provides the fluorescence emission "on demand," since it allows to switch the signal on and off, by washing out non-covalently associated fluorogens [82], as implemented by several groups for the detection of DNA repair enzymes [83][84][85][86]. On the other hand, covalently interacting tags such as HaloTags, SNAP-tags or CLIP-tags offer higher quantum yield and photostability.…”

Recent Advances in Nucleic Acid Targeting Probes and Supramolecular Constructs Based on Pyrene-Modified Oligonucleotides

“…The introduction of non-natural fluorescent monomer Y ( Figure 29 ) into the oligonucleotide substrate of different enzyme bases was introduced by Kool and colleagues. These modified substrates were used to study the activity of a human oxidative enzyme (ALKBH3) [ 166 ], an 8-oxoguanine glycosylase 1 (OGG1) [ 167 ], a 3′-exonuclease (ExoT, RNAse T), a 5′-exonuclease (single-stranded-DNA-specific 5′-exonuclease RecJf), a single-strand endonuclease (nuclease S1) [ 168 ], and bacterial [ 169 , 170 ] and mammalian uracil DNA glycosylase (UDG) [ 170 ].…”