A sensitive and selective mutation detection strategy based on non-canonical DNA structure preference of endonuclease IV
A sensitive and selective mutation detection strategy based on non-canonical DNA structure preference of endonuclease IV
复制标题
基于核酸内切酶IV非典型DNA结构偏好的灵敏选择性突变检测策略
DOI：
10.1016/j.snb.2022.131575
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发表时间：
2022-02-18
影响因子：
8.4
通讯作者：
Wu，Tongbo
中科院分区：
文献类型：
--
作者：
Yuan，Wenqian;Zhang，Zhen;Wu，Tongbo
核酸内切酶IV（Endo IV）因其水解双链DNA（dsDNA）中的脱嘌呤/脱嘧啶位点（AP位点）的特性而成为生化研究领域广泛使用的工具酶。然而，其对具有非典型结构的底物的水解活性尚未被深入研究。本文研究了Endo IV对AP-链和3′-端位于AP位点不同位置的部分互补链形成的dsDNA底物的水解活性。我们发现AP位点周围的单个核苷酸变化可以在某些特定结构中诱导对切割Endo IV的明显减速效应。利用这一特性，我们提出了一种灵敏、选择性高的单核苷酸变异（SNV）检测策略，无需复杂的探针设计、辅助序列和实验条件限制。此外，λ核酸外切酶可以直接引入到系统中，以回收靶链并提高检测能力。对临床相关SNV的检测限达到0.05%突变丰度。我们的检测策略拓展了Endo IV的性质和应用范围，并启发我们从dsDNA底物结构的角度检测SNV。
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.