Abstract：
BACKGROUND: Human 8-oxoG DNA glycosylase 1 (hOGG1) is one of the important members of DNA glycosylase for Base excision repair (BER), the abnormal activity of which can lead to the failure of BER and the appearance of various diseases, such as breast cancer, bladder cancer, Parkinson\'s disease and lung cancer. Therefore, it is important to detect the activity of hOGG1. However, traditional detection methods suffer from time consuming, complicated operation, high false positive results and low sensitivity. Thus, it remains a challenge to develop simple and sensitive hOGG1 analysis strategies to facilitate early diagnosis and treatment of the relative disease.
RESULTS: A target-induced rolling circle amplification (TIRCA) strategy for label-free fluorescence detection of hOGG1 activity was proposed with high sensitivity and specificity. The TIRCA strategy was constructed by a hairpin probe (HP) containing 8-oxoG site and a primer probe (PP). In the presence of hOGG1, the HP transformed into dumbbell DNA probe (DDP) after the 8-oxoG site of which was removed. Then the DDP formed closed circular dumbbell probe (CCDP) by ligase. CCDP could be used as amplification template of RCA to trigger RCA. The RCA products containing repeated G4 sequences could combine with ThT to produce enhanced fluorescence, achieving label-free fluorescence sensing of hOGG1. Given the high amplification efficiency of RCA and the high fluorescence quantum yield of the G4/ThT, the proposed TIRCA achieved highly sensitive measurement of hOGG1 activity with a detection limit of 0.00143 U/mL. The TIRCA strategy also exhibited excellent specificity for hOGG1 analysis over other interference enzymes.
CONCLUSIONS: This novel TIRCA strategy demonstrates high sensitivity and high specificity for the detection of hOGG1, which has also been successfully used for the screening of inhibitors and the analysis of hOGG1 in real samples. We believe that this TIRCA strategy provides new insight into the use of the isothermal nucleic acid amplification as a useful tool for hOGG1 detection and will play an important role in disease early diagnosis and treatment.
RESULTS: A target-induced rolling circle amplification (TIRCA) strategy for label-free fluorescence detection of hOGG1 activity was proposed with high sensitivity and specificity. The TIRCA strategy was constructed by a hairpin probe (HP) containing 8-oxoG site and a primer probe (PP). In the presence of hOGG1, the HP transformed into dumbbell DNA probe (DDP) after the 8-oxoG site of which was removed. Then the DDP formed closed circular dumbbell probe (CCDP) by ligase. CCDP could be used as amplification template of RCA to trigger RCA. The RCA products containing repeated G4 sequences could combine with ThT to produce enhanced fluorescence, achieving label-free fluorescence sensing of hOGG1. Given the high amplification efficiency of RCA and the high fluorescence quantum yield of the G4/ThT, the proposed TIRCA achieved highly sensitive measurement of hOGG1 activity with a detection limit of 0.00143 U/mL. The TIRCA strategy also exhibited excellent specificity for hOGG1 analysis over other interference enzymes.
CONCLUSIONS: This novel TIRCA strategy demonstrates high sensitivity and high specificity for the detection of hOGG1, which has also been successfully used for the screening of inhibitors and the analysis of hOGG1 in real samples. We believe that this TIRCA strategy provides new insight into the use of the isothermal nucleic acid amplification as a useful tool for hOGG1 detection and will play an important role in disease early diagnosis and treatment.
摘要：
背景：人8-oxoGDNA糖基化酶1（hOGG1）是碱基切除修复（BER）的DNA糖基化酶的重要成员之一，其异常活动可能导致BER的失败和各种疾病的出现，比如乳腺癌，膀胱癌,帕金森病和肺癌。因此,重要的是检测hOGG1的活性。然而,传统的检测方法耗时，复杂的操作,假阳性结果高且灵敏度低。因此，开发简单而敏感的hOGG1分析策略以促进相关疾病的早期诊断和治疗仍然是一个挑战.
结果：提出了一种用于无标记荧光检测hOGG1活性的靶标诱导滚环扩增（TIRCA）策略，具有高灵敏度和特异性。TIRCA策略由含有8-oxoG位点的发夹探针(HP)和引物探针(PP)构建。在hOGG1的存在下,在除去其8-oxoG位点后,HP转化到哑铃DNA探针(DDP)中。然后DDP通过连接酶形成封闭的圆形哑铃探针(CCDP)。CCDP可作为RCA的扩增模板触发RCA。含有重复G4序列的RCA产物可以与ThT结合产生增强的荧光，实现hOGG1的无标记荧光传感。鉴于RCA的高扩增效率和G4/ThT的高荧光量子产率,提出的TIRCA实现了对hOGG1活性的高灵敏度测量，检出限为0.00143U/mL。TIRCA策略对hOGG1分析也表现出优于其他干扰酶的优异特异性。
结论：这种新的TIRCA策略显示了检测hOGG1的高灵敏度和高特异性，其也已成功用于筛选抑制剂和分析实际样品中的hOGG1。我们认为，这种TIRCA策略为等温核酸扩增作为hOGG1检测的有用工具的使用提供了新的见解，并将在疾病的早期诊断和治疗中发挥重要作用。
结果：提出了一种用于无标记荧光检测hOGG1活性的靶标诱导滚环扩增（TIRCA）策略，具有高灵敏度和特异性。TIRCA策略由含有8-oxoG位点的发夹探针(HP)和引物探针(PP)构建。在hOGG1的存在下,在除去其8-oxoG位点后,HP转化到哑铃DNA探针(DDP)中。然后DDP通过连接酶形成封闭的圆形哑铃探针(CCDP)。CCDP可作为RCA的扩增模板触发RCA。含有重复G4序列的RCA产物可以与ThT结合产生增强的荧光，实现hOGG1的无标记荧光传感。鉴于RCA的高扩增效率和G4/ThT的高荧光量子产率,提出的TIRCA实现了对hOGG1活性的高灵敏度测量，检出限为0.00143U/mL。TIRCA策略对hOGG1分析也表现出优于其他干扰酶的优异特异性。
结论：这种新的TIRCA策略显示了检测hOGG1的高灵敏度和高特异性，其也已成功用于筛选抑制剂和分析实际样品中的hOGG1。我们认为，这种TIRCA策略为等温核酸扩增作为hOGG1检测的有用工具的使用提供了新的见解，并将在疾病的早期诊断和治疗中发挥重要作用。
