Construction of Bioluminescent Sensors for Label-Free, Template- Free, Separation-Free, and Sequence-Independent Detection of both Clustered and Isolated Damage in Genomic DNA

发布时间：2024-01-19 点击次数：

• 关键字：BASE EXCISION-REPAIR; SHRIMP PANDALUS-BOREALIS; ALKALINE-PHOSPHATASE; 8-OXOGUANINE; TEMPERATURE; COMPLEXES; CHEMISTRY; PROTEIN; ENZYME; SITES
• 摘要：DNA damage induced by endogenous/exogenous factors may cause various diseases, and the genomic DNA damage has become an important biomarker for clinical diagnosis and risk assessment, but it remains a great challenge to accurately quantify both clustered and isolated damage because of their random locations, large diversity, and low abundance. Herein, we demonstrate the development of bioluminescent sensors for label-free, template-free, separation-free, and sequence-independent detection of both clustered and isolated damage in genomic DNA based on the base-excision repair (BER) pathway and terminal transferase (TdT)-initiated template-free isothermal cyclic amplification. The damaged bases are cleaved by DNA glycosylase to generate a new 3 '-OH terminus, and subsequently, TdT catalyzes the repeated incorporation of dTTPs into the 3 '-OH terminus to produce poly-T structures which can hybridize with the signal probe to form a poly-T sequence/signal probe duplex. Under the lambda exonuclease hydrolysis, a large number of adenosine monophosphate (AMP) molecules are produced to generate a high bioluminescence signal through the cyclic interconversion of AMP-adenosine triphosphate (ATP)-AMP in the presence of luciferin and firefly luciferase. Moreover, the introduction of APE1-induced cyclic cleavage signal amplification can greatly improve the detection sensitivity. The proposed strategy can detect both clustered and isolated damage in genomic DNA with extremely high sensitivity and excellent specificity, and it can even distinguish 0.001% DNA damage in the mixture. Importantly, it can detect the cellular DNA damage with a detection limit of 0.011 ng and further extend to measure various DNA damage with the integration of appropriate DNA repair enzymes.
• 卷号：94
• 期号：42
• 是否译文：否