**8. Assays for DNA Glycosylase Activity**

Most basic research on DNA glycosylases was and is still done using radioactively labeled oligonucleotides and analyzing substrates and product by gel electrophoresis. While this assay offers the highest sensitivity, it is labor-intensive, not easily scalable, and inconvenient when screening pharmacological libraries. In the past years, a number of fluorescence-based assays to follow glycosylase activities appeared, some of them coupled with signal amplification to increase the sensitivity.

The first attempts to utilize fluorescent labels for detecting DNA glycosylase activities in a homogeneous mode were based on changes in the signal from a fluorophore incorporated next to a lesion upon the eversion or the excision of the damaged base [232,233] (Figure 2A). Although this approach has been used for inhibitor screening [75], it is not very sensitive, and fluorophores adjacent to a lesion may even inhibit the measured activity. Molecular beacon substrates developed later consist of an oligonucleotide hairpin or a duplex bearing a fluorophore and a quencher at its termini (Figure 2B). Such substrates allow measuring DNA glycosylase activities both in vitro and in living cells [76,234–237] and have been used in glycosylase inhibitor library screening [73,76]. In this case, the glycosylase must be bifunctional to nick the substrate, or else an AP endonuclease has to be present in the assay. Several types of arrays or beads with immobilized damaged oligonucleotides have been reported, in which only the damaged strand is labeled, and the cleavage produces short DNA fragments that can be washed off [238–242]. While such assays are not homogeneous, they are well suited for multiplexing and parallel screening. Fluorophores can also be incorporated into double-stranded DNA in situ through base excision, AP site cleavage, and gap filling by DNA polymerase β with a labeled dNTP [243].

**Figure 2.** Schemes of several fluorescence-based approaches to DNA glycosylase detection. (**A**), fluorescent reporters adjacent to the lesion; (**B**), molecular beacons with a fluorophore–quencher pair; (**C**), T7 exonuclease-assisted signal amplification due to cyclic degradation of a probe bearing a fluorophore–quencher pair.

An interesting approach was suggested that employs a DNAzyme inactivated by a strategically placed U residue, and the excision by uracil-DNA glycosylase reactivates the DNAzyme, which cleaves a fluorescent substrate [244]. However, it cannot be applied to other glycosylases that require double-stranded substrates. In a more advanced version, base excision generates a specifically folded quadruplex, which forms a fluorescent complex with quadruplex-selective ligands [245–247].

The most sensitive fluorescent assays rely on the formation of a nick after the cleavage by a DNA glycosylase (either bifunctional or coupled with an AP endonuclease), followed by signal amplification. The amplification may be exonuclease-assisted linear isothermal, in which a beacon is annealed and degraded by a double-stranded specific exonuclease in repeated cycles [248–250] (Figure 2C). Alternatively, the signal may be enhanced by rolling-circle amplification, using any suitable assay to detect the newly synthesized DNA [251–254]. Finally, nick formation can serve as a starting point for exponential isothermal amplification [255,256] or cascade hybridization [257].
