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Base excision repair (BER) is vital for maintaining the integrity of the genome under oxidative damage. DNA glycosylase initiates the BER pathway recognizes and excises the mismatched substrate base leading to the apurinic/apyrimidinic site generation, and simultaneously breaks the single-strand DNA. As the aberrant activity of DNA glycosylase is associated with numerous diseases, including cancer, immunodeficiency, and atherosclerosis, the detection of DNA glycosylase is significant from bench to bedside. In this review, we summarized novel DNA strategies in the past five years for DNA glycosylase activity detection, which are classified into fluorescence, colorimetric, electrochemical strategies, etc. We also highlight the current limitations and look into the future of DNA glycosylase activity monitoring.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulated diverse cellular processes including differentiation, proliferation, apoptosis, metabolism and signal transduction pathways. An increasing number of data suggested that miRNA-21 could be identified as diagnostic and therapeutic biomarker for breast cancer. Meanwhile, inhibiting the function of miRNA-21, resulting in cells growth inhibition and apoptotic cells death. To realize miRNA-21detection and inhibition to diagnostic and therapeutic breast cancer cells, we developed gold nanoparticle-based 2'-O-methyl modified DNA probes (AuNP-2'-OMe-DNA probes) for diagnostic and therapeutic breast cancer. Gold nanoparticles were functionalized with chemically modified miRNA-21 inhibitor to suppress the function of miRNA-21 for the therapeutic breast cancer, at the same time, fluorophore-labeled DNA molecules were hybridized with antimiRNA-21 for diagnostic breast cancer. The results showed that the 2'-O-methyl modified DNA can improve stability, increase binding affinity to target strands and enhance the therapeutic effects. The experimental results also demonstrated that antimiR-21 were efficiently introduced into the cells and knocked down miRNA-21 to inhibit its function, leading to growth inhibition and apoptotic cells death. We prospected that chemically modified miRNA-21 inhibitor based on gold nanoparticles would be as a promising diagnostic and therapeutic platform for breast cancer clinically.

In this paper, we describe a novel, label-free and nicking enzyme-assisted fluorescence signal amplification strategy that demonstrates to be cost efficient, sensitive, and unique for assaying the RNase H activity and inhibition based on G-quadruplex formation using a thioflavin T (ThT) dye. This novel assay method is able to detect RNase H with a detection limit of 0.03 U /mL and further exhibits a good linearity R2 = 0.9923 at a concentration range of 0.03-1 U/mL under optimized conditions. Moreover, the inhibition effect of gentamycin on the RNase H activity is also studied. This strategy provides a potential tool for the biochemical enzyme analysis and inhibitor screening.

Herein, a turn-on fluorescence assay was introduced for alkaline phosphatase (ALP) detection based on ThT/G-quadruplex system. The basis of the method is that chelation of guanine bases at the binding sites by Ag+ blocks G-quadruplex formation and decreases the fluorescence intensity sharply. In the presence of ALP, ascorbic acid 2-phosphate (AAP) is hydrolyzed to form ascorbic acid (AA) which in turn reduces Ag+ to Ag0. As a result, the blockage ability of Ag+ is disrupted which augments the fluorescence intensity and relies on the concentration of ALP. Under the optimized parameters (500 nM DNA probe; 6 μM Ag+; 1 mM AAP; 30 min for Ag+ and DNA probe reaction time), fluorescence intensity correlates linear range between 1 and 100 U/L of ALP concentration with the detection limit of 0.503 U/L. In the inhibition assay, 50% of ALP inhibition is caused by Na3VO4 with a concentration of 0.254 mM. Furthermore, the assay was used to detect ALP activity in human serum samples in which the results were significant. Above all, the proposed strategy is potential, facile, and sensitive for analyzing ALP activity and screening ALP inhibitor.

We have developed a new methodology for fluorescence turn-on detection of uracil DNA glycosylase (UDG) activity based on G-quadruplex formation using a thioflavin T probe. In the presence of UDG, it catalyzed the hydrolysis of the uracil bases in the duplex DNA, resulting in the dissociation of the duplex DNA owing to their low melting temperature. Then, the probe DNA can be recognized quickly by the ThT dye and resulting in an increase in fluorescence. This approach is highly selective and sensitive with a detection limit of 0.01 U/mL. It is simple and cost effective without requirement of labeling with a fluorophore-quencher pair. This new method could be used to evaluate the inhibition effect of 5-fluorouracil on UDG activity, and become a useful tool in biomedical research.

A sensitive method for rapid angiogenin (Ang) detection based on fluorescence resonance energy transfer (FRET) has been described. A dual-labeled probe based on high affinity aptamer for Ang was constructed. As donor and acceptor, 6-carboxyfluorescein (FAM) and 6-carboxy-tetramethylrhodamine (TMR) were labeled at 5'- and 3'-termini of the aptamer probe, respectively. The dual-labeled probe showed obvious fluorescence changes due to the specific binding between aptamer and Ang. By monitoring the fluorescence intensity of donor and acceptor, quantitative Ang detection could be achieved. This assay is highly specific and sensitive, with a detection limit of 2.0 x 10(-10) mol L(-1) and a linear range of 5.0 x 10(-10) to 4.0 x 10(-8) mol L(-1) Ang. Ang in serum samples of health and lung cancer were also detected.

Traditional methods to assay enzymatic cleavage of DNA are discontinuous, time-consuming and laborious. Here, we report a new approach for real-time monitoring of double-stranded DNA cleavage by restriction endonuclease based on nucleic acid ligation using molecular beacon. Upon cleavage of DNA, the cleavage product can be ligated by DNA ligase, which results in a fluorescence enhancement of the molecular beacon. This method permits real-time monitoring of DNA cleavage and makes it easy to characterize the activity of restriction endonuclease and to study the cleavage reaction kinetics.