A novel one-pot fluorescence tagging and depyrimidination strategy for quantification of global DNA methylation
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 m...
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Published in | Analytica chimica acta Vol. 1239; p. 340636 |
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Main Authors | , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
Netherlands
Elsevier B.V
25.01.2023
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Subjects | |
Online Access | Get full text |
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Summary: | 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.
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•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. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Present Address: School of Chemistry and Chemical Engineering, Frontiers Science Center for Transformative Molecules and National Center for Translational Medicine, Shanghai Jiao Tong University, Shanghai, 200240, China |
ISSN: | 0003-2670 1873-4324 |
DOI: | 10.1016/j.aca.2022.340636 |