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.

Excision Repair-Initiated Enzyme-Assisted Bicyclic Cascade Signal Amplification for Ultrasensitive Detection of Uracil-DNA Glycosylase

As an essential DNA repair enzyme, apurinic/apyrimidinic endonuclease 1 (APE1) is overexpressed in most human cancers and is identified as a cancer diagnostic and predictive biomarker for cancer ri...

Graphene Quantum Dot-Based Nanocomposites for Diagnosing Cancer Biomarker APE1 in Living Cells.

Nicking enzyme-assisted amplification (NEAA) technology and its applications: A review.

Human apurinic/apyrimidinic endonuclease 1 (APE1) is an emerging target for sensitizing tumor cells to radiation and chemotherapy due to its critical function in DNA repair and reduction-oxidation regulation. Thus, accurate detection and intracellular imaging of APE1 hold great promise for drug screening, cancer diagnosis and prognosis evaluation. Here, a DNAzyme modified tetrahedral DNA nanostructure (DZ-TDN) and molecular beacon (MB) incorporated biosensing system was developed to reliably monitor intracellular APE1 activity. DZ-TDN was self-assembled from four single-stranded DNAs, one of which contained an extrude DNAzyme sequence on the 5′-end. At normal status, DNAzyme activity was plugged by performing a duplex with a blocker strand containing an AP site. The digestion of APE1 on this site can restore DNAzyme activity, which consequently trigger MB cleavage recycle to amplify detection signal. The results indicated that the proposed fluorescence method showed a linear response ranged for APE1 from 0.01 to 1 U mL−1 with a limit of detection (LOD) of 0.01 U mL−1 (S/N = 3). Moreover, the strategy was successfully used for effectors screening and intracellular imaging of APE1 with high reliability.

Activity assay and intracellular imaging of APE1 assisted with tetrahedral DNA nanostructure modified-dnazyme and molecular beacon

Human apurinic/apyrimidinic endonuclease 1 (APE1) plays a crucial role in DNA repair and gene regulation. The abnormal variations of the concentration of APE1 in the human blood and tissue cells are highly correlated to various diseases. Thus, APE1 can be used as a biomarker to aid clinical diagnosis of diseases. Detection of APE1 activity with high sensitivity and selectivity simultaneously is difficult to achieve. Here we provide a DNA structure-mediated fluorescent biosensor to solve the above problem. Upon the existence of APE1, the apurinic/apyrimidinic site will be cleaved, and the terminal structure of the hairpin DNA substrate will change. Then the terminal deoxynucleotidyl transferase and endonuclease IV could provide dual fluorescent signal amplification. Through the ingenious structure design of the biosensor, we have improved the reactivity of APE1 and minimized the interference of other enzymes. The results indicate that the detection limit for APE1 is as low as 1.7 × 10−6 U/mL. The biosensor has been successfully applied to the detection of APE1 in real biological samples and the screening of APE1 inhibitors.

A DNA structure-mediated fluorescent biosensor for apurinic/apyrimidinic endonuclease 1 activity detection with ultra-high sensitivity and selectivity

A label-free fluorescence assay for the detection of apurinic/apyrimidinic endonuclease 1 activity was developed based on polymerase and nicking endonuclease co-assisted isothermal amplification and G-quadruplex/ligand interaction. Through signal amplification, this method displays high sensitivity with a detection limit of 0.006 U mL−1.

Sensitive and label-free fluorescence detection of apurinic/apyrimidinic endonuclease 1 activity based on isothermal amplified-generation of G-quadruplex

We introduced a simple, fast and label free fluorescent assay for uracil-DNA glycosylase activity based on the signal amplification of exonuclease I. This method exhibits a high sensitivity for UDG activity assay with a low detection limit of 0.0070 U mL−1.

A label free fluorescent assay for uracil-DNA glycosylase activity based on the signal amplification of exonuclease I

Apurinic/apyrimidinic endonuclease 1 (APE1) plays crucial role in DNA repair, which cleaves the abasic site (AP site) to facilitate the DNA repair process. APE1 is overexpressed in several cancers, and it has been demonstrated to be a promising target for anticancer drugs. Thus, development of a method for the detection of APE1 activity and screening of its inhibitors is important and highly desirable for cancer diagnosis and therapy. In this study, we report a simple and label-free fluorescence detection method for APE1 activity using an AP site-binding dye (2-amino-5,6,7-trimethyl-1,8-naphthyridine, ATMND) as a signal indicator. The fluorescence of ATMND was quenched after inserting ATMND into the AP site with a C base at the opposite position. The APE1-catalyzed cleavage of the AP site resulted in the release of the bound ATMND, and the quenched fluorescence of ATMND recovered. The label-free strategy makes this method convenient and easy to manipulate. The proposed method exhibits a good sensitivity, and we have also employed it to evaluate the inhibition effect of a drug on the APE1 activity. This approach holds potential applications in APE1-related cancer diagnosis and anticancer drug screening.

Simple label-free fluorescence detection of apurinic/apyrimidinic endonuclease 1 activity and its inhibitor using the abasic site-binding fluorophore

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Sensitive and label-free fluorescence detection of apurinic/apyrimidinic endonuclease 1 activity based on isothermal amplified-generation of G-quadruplex

A label free fluorescent assay for uracil-DNA glycosylase activity based on the signal amplification of exonuclease I

Simple label-free fluorescence detection of apurinic/apyrimidinic endonuclease 1 activity and its inhibitor using the abasic site-binding fluorophore