Abstract：
BACKGROUND: As a core enzyme in the base excision repair system, uracil DNA glycosylase (UDG) is indispensable in maintaining genomic integrity and normal cell cycles. Its abnormal activity intervenes in cancers and neurodegerative diseases. Previous UDG assays based on isothermal amplification and Clustered Regularly Interspaced Short Palindromic Repeats/Cas (CRISPR/Cas) system were fine in sensitivity, but exposed to complications in assay flow, time, and probe design. After isothermal amplification, a CRISPR/Cas reagent should be separately added with extra manual steps and its guide RNA (gRNA) should be designed, considering the presence of protospacer adjacent motif (PAM) site.
RESULTS: We herein describe a UDG-REtarded CRISPR Amplification assay, termed \'URECA\'. In URECA, isothermal nucleic acid (NA) amplification and CRISPR/Cas12a system were tightly combined to constitute a one-pot, isothermal CRISPR amplification system. Isothermal NA amplification for a UDG substrate (US) with uracil (U) bases was designed to activate and boost CRISPR/Cas12a reaction. Such scheme enabled us to envision that UDG would halt the isothermal CRISPR amplification reaction by excising U bases and messing up the US. Based on this principle, the assay detected the UDG activity down to 9.17 x 10-4 U/mL in 50 min. With URECA, we fulfilled the recovery test of UDG activities in plasma and urine with high precision and reproducibility and reliably determined UDG activities in cell extracts. Also, we verified its capability to screen candidate UDG inhibitors, showing its potentials in practical application as well as drug discovery.
CONCLUSIONS: URECA offers further merits: i) the assay is seamless. Following target recognition, the reactions proceed in one-step without any intervening steps, ii) probe design is simple. Unlike the conventional CRISPR/Cas12a-based assays, URECA does not consider the PAM site in probe design as Cas12a activation relies on instantaneous gRNA binding to single-stranded DNA strands. By rationally designing an enzyme substrate probe to be specific to other enzymes, while keeping a role as a template for isothermal CRISPR amplification, the detection principle of URECA will be expanded to devise biosensors for various enzymes of biological, clinical significance.
RESULTS: We herein describe a UDG-REtarded CRISPR Amplification assay, termed \'URECA\'. In URECA, isothermal nucleic acid (NA) amplification and CRISPR/Cas12a system were tightly combined to constitute a one-pot, isothermal CRISPR amplification system. Isothermal NA amplification for a UDG substrate (US) with uracil (U) bases was designed to activate and boost CRISPR/Cas12a reaction. Such scheme enabled us to envision that UDG would halt the isothermal CRISPR amplification reaction by excising U bases and messing up the US. Based on this principle, the assay detected the UDG activity down to 9.17 x 10-4 U/mL in 50 min. With URECA, we fulfilled the recovery test of UDG activities in plasma and urine with high precision and reproducibility and reliably determined UDG activities in cell extracts. Also, we verified its capability to screen candidate UDG inhibitors, showing its potentials in practical application as well as drug discovery.
CONCLUSIONS: URECA offers further merits: i) the assay is seamless. Following target recognition, the reactions proceed in one-step without any intervening steps, ii) probe design is simple. Unlike the conventional CRISPR/Cas12a-based assays, URECA does not consider the PAM site in probe design as Cas12a activation relies on instantaneous gRNA binding to single-stranded DNA strands. By rationally designing an enzyme substrate probe to be specific to other enzymes, while keeping a role as a template for isothermal CRISPR amplification, the detection principle of URECA will be expanded to devise biosensors for various enzymes of biological, clinical significance.
摘要：
背景：作为碱基切除修复系统中的核心酶，尿嘧啶DNA糖基化酶（UDG）在维持基因组完整性和正常细胞周期中是必不可少的。其异常活性干预癌症和神经变性疾病。以前的基于等温扩增和成簇定期间隔短回文重复/Cas（CRISPR/Cas）系统的UDG测定灵敏度很好，但是在分析流程中遇到了并发症，时间,和探头设计。等温扩增后,CRISPR/Cas试剂应单独添加额外的手动步骤，并设计其指导RNA（gRNA），考虑到原型间隔区相邻基序(PAM)位点的存在。
结果：我们在此描述了UDG-retardedCRISPR扩增测定，称为“URECA”。在乌雷卡,等温核酸(NA)扩增与CRISPR/Cas12a系统紧密结合构成一锅法,等温CRISPR扩增系统。设计具有尿嘧啶(U)碱基的UDG底物(US)的等温NA扩增以激活和促进CRISPR/Cas12a反应。这样的方案使我们能够设想UDG将通过切除U碱基并弄乱美国来停止等温CRISPR扩增反应。基于这个原则,该试验检测到UDG活性在50分钟内降至9.17×10-4U/mL。有了乌雷卡,我们完成了血浆和尿液中UDG活性的回收率测试，具有很高的精密度和重现性，并且可靠地测定了细胞提取物中的UDG活性。此外，我们验证了它筛选候选UDG抑制剂的能力，显示其在实际应用和药物发现中的潜力。
结论:URECA提供了进一步的优点:i)该测定是无缝的。在目标识别之后,反应一步进行，没有任何中间步骤，ii)探头设计简单。与传统的基于CRISPR/Cas12a的检测方法不同，URECA在探针设计中不考虑PAM位点,因为Cas12a激活依赖于瞬时gRNA与单链DNA链的结合。通过合理设计对其他酶具有特异性的酶底物探针，同时保持作为等温CRISPR扩增模板的作用，URECA的检测原理将扩展到为各种生物酶设计生物传感器，临床意义。
结果：我们在此描述了UDG-retardedCRISPR扩增测定，称为“URECA”。在乌雷卡,等温核酸(NA)扩增与CRISPR/Cas12a系统紧密结合构成一锅法,等温CRISPR扩增系统。设计具有尿嘧啶(U)碱基的UDG底物(US)的等温NA扩增以激活和促进CRISPR/Cas12a反应。这样的方案使我们能够设想UDG将通过切除U碱基并弄乱美国来停止等温CRISPR扩增反应。基于这个原则,该试验检测到UDG活性在50分钟内降至9.17×10-4U/mL。有了乌雷卡,我们完成了血浆和尿液中UDG活性的回收率测试，具有很高的精密度和重现性，并且可靠地测定了细胞提取物中的UDG活性。此外，我们验证了它筛选候选UDG抑制剂的能力，显示其在实际应用和药物发现中的潜力。
结论:URECA提供了进一步的优点:i)该测定是无缝的。在目标识别之后,反应一步进行，没有任何中间步骤，ii)探头设计简单。与传统的基于CRISPR/Cas12a的检测方法不同，URECA在探针设计中不考虑PAM位点,因为Cas12a激活依赖于瞬时gRNA与单链DNA链的结合。通过合理设计对其他酶具有特异性的酶底物探针，同时保持作为等温CRISPR扩增模板的作用，URECA的检测原理将扩展到为各种生物酶设计生物传感器，临床意义。
