Label‐Free Quantification of 5‐Azacytidines Directly in the Genome

Abstract

Azacytidines (AzaC and AzadC) are clinically relevant pharmaceuticals that operate at the epigenetic level. They are integrated into the genome as antimetabolites to block DNA methylation events. This leads to a reduction of the 5‐methyl‐2′‐deoxycytidine (m5dC) level in the genome, which can activate epigenetically silenced genes. Because of the inherent chemical instability of Aza(d)Cs, their incorporation levels in DNA and RNA are difficult to determine, which hinders correlation of therapeutic effects with incorporation and removal processes. Existing methods involve radioactive labeling and are therefore unsuitable to monitor levels from patients. We report here a new direct chemical method that allows absolute quantification of the levels of incorporated AzaC and AzadC in both RNA and DNA. Furthermore, it clarifies that Aza(d)C accumulates to high levels (up to 12.9 million bases per genome). Although RNA‐based antimetabolites are often 2′‐deoxygenated in vivo and incorporated into DNA, for AzaC we see only limited incorporation into DNA. It accumulates predominantly in RNA where it, however, only leads to insignificant demethylation.

Comprehensive comparison between azacytidine and decitabine treatment in an acute myeloid leukemia cell line

“…First, we checked by ultra-HPLC triple quadrupole mass spectrometry (UHPLC-QQQ-MS) whether the global mdC level changed upon AzaC and AzadC treatment as expected. In accordance with previously published results [7], mdC levels decreased after exposure to AzaC and AzadC for 72 h (Fig. 1a).…”

Influencing Epigenetic Information with a Hydrolytically Stable Carbocyclic 5‐Aza‐2′‐deoxycytidine

“…Parallel to the quantification of mdC and hmdC, we also quantified to which extent cAzadC itself was incorporated into the genome of the mESCs.D etection of AzadC in the genome of treated cells is only possible after treatment of the DNAw ith NaBH 4 .A pplication of NaBH 4 reduces the C(5) = C(6) double bond, which stabilizes the compound so that its quantification becomes possible. [19,23] To our delight, we noted that the stability of cAzadC allowed its quantification without this pre-treatment. We also noted that the applied enzymatic digestion protocol allowed digestion of genomic DNA(gDNA) even in the presence of large amounts of cAzadC.T aken together,q uantification of cAzadC by UHPLC-MS 2 using an external calibration curve ( Figure S2) was possible in parallel with quantification of canonical and epigenetic bases.…”

Influencing Epigenetic Information with a Hydrolytically Stable Carbocyclic 5‐Aza‐2′‐deoxycytidine

“…At the higher concentration of 5 μ m cAzadC, the level increased 3‐fold to 1.7×10 −3 cAzadC per dN and consequently to more than 8 million integrated cAzadC nucleotides per genome. Compared to the incorporation of AzadC, which reaches 1.2×10 −3 AzadC per dN, when applied with 1 μ m , the levels of cAzadC reached about a third of this level. The data clearly show that the carbocyclic version of AzadC (cAzadC) is incorporated and that it reaches in the genome finally comparable levels at 5 μ m concentration.…”