Integration of Enzymatic Labeling with Single-Molecule Detection for Sensitive Quantification of Diverse DNA Damages.

Yan Zhang, Ruo-Nan Hua, Chun-Yang Zhang

Analytical chemistry 2020 Apr 07

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DNA damage plays an important role in the regulation of gene expression and disease processes. The accurate measurement of DNA damage is essential to the discovery of potential disease biomarkers for risk assessment, early clinical diagnosis, and therapy monitoring. However, the low abundance, random location in genomic elements, diversity, and the incapability to specifically amplify the DNA damages hinder the accurate quantification of various DNA damages within human genomes. Herein, we demonstrate the integration of enzymatic labeling with single-molecule detection for sensitive quantification of diverse DNA damages. A significant advantage of our method is that only the damaged base-containing DNA sequence can be labeled by the biotin-conjugated deoxynucleotide triphosphate (biotin-dNTP) and separated from the normal DNAs, which greatly improves the detection specificity. In addition, high sensitivity can be achieved by the terminal deoxynucleotidyl transferase (TdT)-induced polymerization of multiple Alexa Fluor 488-labeled-deoxyuridine triphosphates (AF488-dUTPs) and the introduction of single-molecule detection. This method can measure DNA damage with a detection limit as low as 1.1 × 10-16 M, and it can distinguish DNA damage at low abundance down to 1.3 × 10-4%. Importantly, it can provide information about the occurrence of DNA damage in a specific gene and ascertain the DNA damage level in different cancer cell lines, offering a new approach for studying the physiological function of various DNA damages in human diseases.

Citation

Yan Zhang, Ruo-Nan Hua, Chun-Yang Zhang. Integration of Enzymatic Labeling with Single-Molecule Detection for Sensitive Quantification of Diverse DNA Damages. Analytical chemistry. 2020 Apr 07;92(7):4700-4706