A sensitive and selective mutation detection strategy based on non-canonical DNA structure preference of endonuclease IV

Endonuclease IV (Endo IV) is a widely used tool enzyme in biochemical research fields due to its properties in hydrolyzing the apurinic/apyrimidinic site (AP site) in the double-strand DNA (dsDNA). However, its hydrolysis activity towards the substrate with an atypical structure has not been deeply investigated yet. Here, the hydrolysis activity of Endo IV to the dsDNA substrate formed by AP-strand and partially complementary strands whose 3′-end located at different positions of the AP site was studied in detail. We found that just a single nucleotide change around the AP site could induce an apparent deceleration effect on the cleavage Endo IV in some specific structures. Benefiting from the above property, we proposed a sensitive and selective single-nucleotide variation (SNV) detection strategy without complicated probe design, auxiliary sequences or restricted experimental conditions. Furthermore, lambda exonuclease could be directly introduced to the system to recycle the target strands and enhance the detection capability. The limit of detection towards clinical-related SNV reached 0.05% mutation abundance. The detection strategy in our work expanded our horizon of properties and applications of Endo IV and inspired us to detect SNV from the perspective of dsDNA substrate structure.