Here we describe a novel endonuclease IV (Endo IV) assay substrate ( Figure 1C ) that mimics the abasic lesions that normally occur in double-stranded DNA ( Figure 1A and B). The first lesion ( Figure 1A ) is a typical abasic (apurinic or apyrimidinic site generated by spontaneous or enzymatic loss of a nucleic acid base. The second lesion ( Figure 1B) is an atypical abasic site appearing as a result of inherent instability of the 3 0 -phosphodiester bond in abasic deoxyribose in lesion 1 or its cleavage by a Class I AP endonuclease. As shown by the arrow, AP endonucleases cleave the phosphodiester linkage at the abasic sites. In the novel Endo IV assay a short probe with a fluorophore linked through a phosphate at the 3 0 end and a short enhancer oligonucleotide generate an artificial, lesion 2 type Endo IV substrate. This arrangement allows the specific and efficient cleavage of the phosphodiester bond of the probe by Endo IV and release of fluorescent dye ( Figure 1C ). The addition of a quencher at the 5 0 end of the probe allows quenching of the fluorescence in the uncleaved probe. The specific cleavage of the phosphate bond and the generation of fluorescence is the basis of the new assay.