ABSTRACT
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.
Subject(s)
DNA/genetics , Single Molecule Imaging , Cell Line , DNA Damage , HumansABSTRACT
DNA damage seriously threats the genomic stability and is linked to mutagenesis, carcinogenesis, and cell death. DNA damage includes the isolated damage and the clustered damages, but few approaches are available for efficient detection of the clustered damage due to its spatial distribution. Herein, we present a single-molecule counting approach with the capability of detecting both the isolated and the clustered damages in genomic DNAs. We employed the repair enzymes to remove the DNA damage and used the terminal deoxynucleotidyl transferase (TdT) to incorporate biotinylated nucleotides and fluorescent nucleotides into the damage sites in a template-independent manner. The number of total oxidative damaged bases is quantified to be 7328-7406 in a single HeLa cell treated with 150 µM H2O2. This method in combination with special repair enzymes can detect a variety of DNA damage in different types of cells, holding great potential for early diagnosis of DNA damage-related human diseases.
Subject(s)
DNA Repair/drug effects , DNA/analysis , Single Molecule Imaging/methods , Staining and Labeling/methods , Biotin/chemistry , Biotinylation , Carbocyanines/chemistry , DNA/genetics , DNA/metabolism , DNA Damage , DNA Glycosylases/chemistry , DNA Glycosylases/metabolism , DNA Nucleotidylexotransferase/chemistry , DNA Nucleotidylexotransferase/metabolism , DNA-(Apurinic or Apyrimidinic Site) Lyase/chemistry , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Deoxyribonucleotides/chemistry , Deoxyribonucleotides/metabolism , HeLa Cells , Humans , Hydrogen Peroxide/pharmacology , Streptavidin/chemistryABSTRACT
We demonstrate for the first time the single-molecule counting of oxidative DNA damage in telomeres from a human cervical carcinoma cell line (HeLa cells). This method exhibits high sensitivity towards oxidative DNA damage with a detection limit as low as 9.3 × 10-17 M and good discrimination capability down to the 0.001% oxidative damage level. Moreover, this method can quantify the number of oxidative damaged bases (34-44) in telomeres in each HeLa cell treated with 1000 µM H2O2.