title: Excision Repair-Initiated Enzyme-Assisted Bicyclic Cascade Signal Amplification for Ultrasensitive Detection of Uracil-DNA Glycosylase.
abstract: Uracil-DNA glycosylase (UDG) is an important base excision repair (BER) enzyme responsible for the repair of uracil-induced DNA lesion and the maintenance of genomic integrity, while the aberrant expression of UDG is associated with a variety of cancers. Thus, the accurate detection of UDG activity is essential to biomedical research and clinical diagnosis. Here, we develop a fluorescent method for ultrasensitive detection of UDG activity using excision repair-initiated enzyme-assisted bicyclic cascade signal amplification. This assay involves (1) UDG-actuated uracil-excision repair, (2) excision repair-initiated nicking enzyme-mediated isothermal exponential amplification, (3) ribonuclease H (RNase H)-induced hydrolysis of signal probes for generating fluorescence signal. The presence of UDG enables the removal of uracil from U·A pairs and generates an apurinic/apyrimidinic (AP) site. Endonuclease IV (Endo IV) subsequently cleaves the AP site, resulting in the break of DNA substrate. The cleaved DNA substrate functions as both a primer and a template to initiate isothermal exponential amplification, producing a large number of triggers. The resultant trigger may selectively hybridize with the signal probe which is modified with FAM and BHQ1, forming a RNA-DNA heterogeneous duplex. The subsequent hydrolysis of RNA-DNA duplex by RNase H leads to the generation of fluorescence signal. This assay exhibits ultrahigh sensitivity with a detection limit of 0.0001 U/mL, and it can even measure UDG activity at the single-cell level. Moreover, this method can be applied for the measurement of kinetic parameters and the screening of inhibitors, thereby providing a powerful tool for DNA repair enzyme-related biomedical research and clinical diagnosis.
authors.affiliation: College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University , Jinan 250014, China.
authors.forename: Li-Juan
authors.initials: LJ
authors.lastname: Wang
authors.affiliation: College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University , Jinan 250014, China.
authors.forename: Ming
authors.initials: M
authors.lastname: Ren
authors.affiliation: Nantou High School Shenzhen , Shenzhen, 518052, China.
authors.forename: Qianyi
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University , Jinan 250014, China.
authors.forename: Bo
authors.identifier: 0000-0002-8712-7025
authors.initials: B
authors.lastname: Tang
authors.affiliation: College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University , Jinan 250014, China.
authors.forename: Chun-Yang
authors.identifier: 0000-0002-8010-1981
authors.initials: CY
authors.lastname: Zhang
mesh_terms: D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004260:DNA Repair; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D021141:Nucleic Acid Amplification Techniques; D013050:Spectrometry, Fluorescence; D014498:Uracil / Q000378:metabolism; D051981:Uracil-DNA Glycosidase / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D014498:Uracil; D004247:DNA; D051981:Uracil-DNA Glycosidase