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Target-mediated hyperbranched amplification for sensitive detection of human alkyladenine DNA glycosylase from HeLa
Lili Wang1, Huige Zhang1, Yi Xie2
1State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China.
Human alkyladenine DNA glycosylase (hAAG) is an important protein enzyme which can specifically recognize and initiate the repair of a variety of alkylated purines and hypoxanthine, and the dysregulation of hAAG activity is associated with various human diseases. Although there are several methods focusing on hAAG detection, they share common defects such as time-consuming protocols, laborious operation or requirement of expensive analytical instruments. Herein, taking advantage of the high amplification efficiency of hyperbranched signal amplification and the low background signals by modifying NH2 at 3' terminus of hairpin substrate and signal probe to prevent the terminal deoxynucleotidyl transferase (TdT)-activated nonspecific amplification, a fluoresence method for sensitive detection of hAAG was established using TdT-activated Endonuclease IV (Endo IV)-assisted hyperbranched signal amplification. This method exhibits high sensitivity with a limit of detection of 0.090 U/mL for pure hAAG and shows a large dynamic range of 3 orders of magnitude from 0.1 to 50 U/mL, and it can be applied for accurate detection of hAAG in complicated HeLa nuclear extract. Moreover, the method can be used for discrimination of hAAG from other DNA glycosylases, holding great potential in hAAG-related biomedical research and clinical diagnosis.
Human alkyladenine DNA glycosylase (hAAG) is an important protein enzyme which can specifically recognize and initiate the repair of a variety of alkylated purines and hypoxanthine, and the dysregulation of hAAG activity is associated with various human diseases. Although there are several methods focusing on hAAG detection, they share common defects such as time-consuming protocols, laborious operation or requirement of expensive analytical instruments. Herein, taking advantage of the high amplification efficiency of hyperbranched signal amplification and the low background signals by modifying NH2 at 3' terminus of hairpin substrate and signal probe to prevent the terminal deoxynucleotidyl transferase (TdT)-activated nonspecific amplification, a fluoresence method for sensitive detection of hAAG was established using TdT-activated Endonuclease IV (Endo IV)-assisted hyperbranched signal amplification. This method exhibits high sensitivity with a limit of detection of 0.090 U/mL for pure hAAG and shows a large dynamic range of 3 orders of magnitude from 0.1 to 50 U/mL, and it can be applied for accurate detection of hAAG in complicated HeLa nuclear extract. Moreover, the method can be used for discrimination of hAAG from other DNA glycosylases, holding great potential in hAAG-related biomedical research and clinical diagnosis.
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