Discrimination between 5-Hydroxymethylcytosine and 5-Methylcytosine in DNA via Selective Electrogenerated Chemiluminescence (ECL) Labeling.
DNA methylation is used to dynamically reprogram cells in the course of early embryonic development in mammals. 5-Hydroxymethylcytosine in DNA (5-hmC-DNA) plays essential roles in the demethylation processes. 5-Methylcytosine in DNA (5-mC-DNA) is oxidized to 5-hmC-DNA by 10-11 translocation proteins, which are relatively high abundance in embryonic stem cells and neurons. A new method was developed herein to quantify 5-hmC-DNA based on selective electrogenerated chemiluminescence (ECL) labeling with the specific oxidation of 5-hmC to 5-fC by KRuO4. A thiolated capture probe (ssDNA, 35-mer) for the target DNA containing 5-hmC was self-assembled on a gold surface. The 5-hmC in the target DNA was selectively transformed to 5-fC via oxidation by KRuO4and then subsequently labeled with N-(4-aminobutyl)-N-ethylisoluminol (ABEI). The ABEI-labeled target DNA was hybridized with the capture probe on the electrode, resulting in a strong ECL emission. An extremely low detection limit of 1.4 × 10-13M was achieved for the detection of 5-hmC-DNA. In addition, this ECL method was useful for the quantification of 5-hmC in serum samples. This work demonstrates that selective 5-hmC oxidation in combination with an inherently sensitive ECL method is a promising tactic for 5-hmC biosensing.
Key Laboratory of Electroanalytical Chemistry of Shaanxi Province, Institute of Analytical Science, Northwest University , Xi'an, 710069, People's Republic of China.
Shangxian
S
Ma
Key Laboratory of Electroanalytical Chemistry of Shaanxi Province, Institute of Analytical Science, Northwest University , Xi'an, 710069, People's Republic of China.
Huiping
H
Sun
Key Laboratory of Electroanalytical Chemistry of Shaanxi Province, Institute of Analytical Science, Northwest University , Xi'an, 710069, People's Republic of China.
Yan
Y
Li
Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University , Xi'an, 710062, People's Republic of China.
Honglan
H
Qi
Key Laboratory of Electroanalytical Chemistry of Shaanxi Province, Institute of Analytical Science, Northwest University , Xi'an, 710069, People's Republic of China.
Jianbin
J
Zheng
D044503:5-Methylcytosine / Q000031:analogs & derivatives* / Q000097:blood / Q000737:chemistry*; D015374:Biosensing Techniques / Q000379:methods*; D003596:Cytosine / Q000031:analogs & derivatives / Q000737:chemistry; D004247:DNA / Q000097:blood* / Q000235:genetics; D015342:DNA Probes; D006801:Humans; D057230:Limit of Detection; D049449:Luminescence; D008165:Luminol / Q000031:analogs & derivatives / Q000737:chemistry; D009693:Nucleic Acid Hybridization; D010084:Oxidation-Reduction; D012428:Ruthenium / Q000737:chemistry
C560973:5-formylcytosine; D015342:DNA Probes; C561085:N-(4-aminobutyl)-N-ethylisoluminol; C011865:5-hydroxymethylcytosine; D008165:Luminol; D044503:5-Methylcytosine; D012428:Ruthenium; D003596:Cytosine; D004247:DNA