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Novel, direct and chemical method of quantifying 5-Azacytidines in the genome


The RNA nucleoside 5-Azacytidine (AzaC) and its DNA analogue 5-Aza-2’-deoxycytidine (Decitabine, AzadC) are epigenetic modulators that are clinically used for the treatment of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). AzaC and AzadC incorporate into RNA and DNA to inhibit DNA methylation, by blocking methyltransferases (Dnmt1 and 3a/3b). The consequent reduction in m5dC levels in the genome can reactivate silenced tumor suppressor genes. The correlation between the incorporation of Aza(d)C compounds and their therapeutic effects and the degree of DNA methylation inhibited will help understand the compounds’ mechanism of action(s). However, due to the biochemical instability of Aza(d)C, direct quantification of incorporation into DNA and RNA is complicated. 

The half-life of Aza(d)C is between 3.5 h and 21 h and thus have low hydrolytic instability. Measuring these compounds within DNA and RNA directly is therefore difficult. This study developed a new method for quantifying the levels of Aza(d)C compounds (from Carbosynth - NA02947 and BA164359) that are incorporated into DNA and RNA, which does not involve radioactive labelling. The method developed is based on mass spectroscopy and enables direct quantification of Aza(d)C compounds, as well as m5(d)C levels.

The developed protocol was tested in DNA, isolated from AzadC-treated cells, which was subsequently treated with NaBH4 (forming H2-AzadC) for stability. The signal for H2-AzadC was detected at increasing intensity in a dose-dependent manner, which was accompanied by a decline in m5dC level. Unlike AzadC, the incorporation of AzaC into DNA was lower, with minor effect on demethylation, and rather accumulates in RNA.

The methods developed in this study can be applied in patients receiving Aza(d)C as their treatment, since it does not involve radioactive labelling. Future studies using this method will be beneficial for understanding resistance to Aza(d)C therapy. 

For further information, please refer to the original paper: Schiffers et al, 2019


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