Recent research reveals that human alkyladenine

Recent research reveals that human alkyladenine DNA glycosylase (hAAG) is an important protein enzyme which can specifically recognize and excise a variety of alkylated purines and deoxyinosine from DNA [12]. hAAG plays pivotal roles in maintaining genomic integrity, and it is involved in bms-690514 regulation, apoptosis [13], and various diseases such as lung cancer [14] and cervical cancer [15]. In particular, hAAG has been shown to induce frameshift mutagenesis and microsatellite instability in human cells [16]. Therefore, the accurate and sensitive detection of hAAG activity is crucial to both biomedical research and clinical diagnosis. To date, only a few approaches are available for hAAG assay. Polyacrylamide gel electrophoresis is considered as the conventional method [17]. However, it suffers from poor sensitivity and involves hazardous radiation and time-consuming protocols. Alternatively, several methods including magnetic nanoparticle-based separation techniques [15] and highly sensitive single-molecule detection [14] have been developed in recent years. Magnetic nanoparticle-based separation method employs the immobilization of fluorescent DNA nanoprobes on magnetic beads, enabling the fluorescent detection of hAAG activity in the supernatant, but it usually needs long analysis time and complicated procedures. The highly sensitive single-molecule detection method is based on the DNA glycosylase mediated cleavage of molecular beacons, but the single-molecule instrument is expensive and difficult to be used prevalently [18]. Thus, it's necessary to develop a simple and sensitive method to detect low-abundance hAAG. To improve the detection sensitivity, several amplification strategies have been introduced, including strand displacement amplification (SDA) [19], [20], rolling circle amplification (RCA) [21], exponential isothermal amplification (EXPAR) [22], [23] and loop-mediated isothermal amplification (LAMP) [24]. Despite the improved sensitivity, the signal amplification approaches usually require complicated probes and templates, accompanied by high background signal due to the involvement of nonspecific amplification [25]. Deoxynucleotidyl transferase (TdT) is an enzyme that can catalyze the repetitive addition of deoxynucleotide triphosphates (dNTPs) to the 3’-OH terminus of a DNA sequence without the requirement of any DNA templates as primers [26], [27] and has been exploited as a powerful tool for bioanalysis [28]. Therefore, it will be of great significance to develop a simple and sensitive method to detect hAAG based on the TdT-mediated polymerization reaction. In this research, we develop a fluorescence method to sensitively detect hAAG on the basis of TdT-activated endonuclease IV (Endo IV)-assisted hyperbranched signal amplification. In comparison with the reported methods for hAAG assay, the method based on the TdT-activated Endo IV-assisted hyperbranched signal amplification has significant advantage of simple reaction process using only two DNA oligonucleotides in a simple ‘‘mix and detection’’ manner, avoiding the involvement of complicated immobilization, separation and washing steps, greatly simplifying the experimental procedure. Moreover, the hAAG activity detection can be finished in a short time (∽100 min). Taking advantage of the high amplification efficiency of hyperbranched signal amplification and the low background signals by modifying NH2 at 3’ termini of hairpin substrate and signal probe to prevent the TdT-activated nonspecific amplification, the proposed method shows good specificity and high sensitivity with a detection limit as low as 0.090 U/mL for pure hAAG and exhibits a large dynamic range of 3 orders of magnitude from 0.1 to 50 U/mL. Moreover, the method can be applied for the quantification the hAAG activity in Hela cells, holding great potential in biomedical research and clinical diagnosis.