A novel one-pot fluorescence tagging and depyrimidination strategy for quantification of global DNA methylation

• Published in: Analytica chimica acta Vol. 1239; p. 340636
• Main Authors: Liao, Xun, Bai, Xiaolin, Wang, Shuguan, Liggins, Christany, Pan, Li, Wang, Meiyuan, Tchounwou, Paul, Mao, Jinghe, Liu, Yi-Ming
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 25.01.2023
• Subjects: 5-Methylcytosine - analysis; Chloroacetaldehyde; Chromatography, High Pressure Liquid - methods; Cytosine; DNA - analysis; DNA Methylation; HCT-116 cells; HPLC-FD analysis; Methylcytosine; Prostatic and breast tissues; Yeast; Chloroacetaldehyde; Methylcytosine; Yeast; Prostatic and breast tissues; HPLC-FD analysis; HCT-116 cells
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/j.aca.2022.340636

DNA methylation is intensively studied in medical science. Current HPLC methods for quantification of global DNA methylation involve digestion of a DNA sample and HPLC determination of both cytosine (C) and 5-methylcytosine (5mC) so that percentage of 5mC in total cytosine can be calculated as DNA methylation level. Herein we report a novel HPLC method based on a one-pot fluorescence tagging and depyrimidination reaction between DNA and chloroacetaldehyde (CAA) for highly sensitive quantification of global DNA methylation. In the one-pot reaction, C and 5mC residues in a DNA sequence react with CAA, forming fluorescent etheno-adducts that are then released from the sequence through depyrimidination. Interestingly, etheno-5mC (ε-5mC) is ∼20 times more fluorescent than ε-C and other ε-nucleobases resulting from the reaction, which greatly facilitates the quantification. Further, due to the tagging-induced increase in structural aromaticity, ε-nucleobases are far more separable by HPLC than intact nucleobases. The proposed HPLC method with fluorescence detection (HPLC-FD) is quick (i.e., < 1h per assay) and highly sensitive with a detection limit of 0.80 nM (or 250 fg on column) for 5mC. Using the method, DNA samples isolated from yeast, HCT-116 cells, and tissues were analyzed. Global DNA methylation was measured to be in the range from 0.35% to 2.23% in the samples analyzed. This sensitive method allowed accurate analyses of minute DNA samples (∼100 ng) isolated from milligrams of tissues. [Display omitted] •The novel analytical strategy is based on a one-pot fluorescence tagging and depyrimidination reaction between DNA and chloroacetaldehyde.•The fluorescence tagging reagent, i.e., chloroacetaldehyde, is advantageous over other tagging reagents because of its zero-fluorescence background.•Separation of nucleobase derivatives is achieved for the first time with a salt free mobile phase and simple isocratic elution.•The HPLC-FD method proposed is far more sensitive and faster than most of the HPLC methods previously reported.