Ultrasensitive Determination of Rare Modified Cytosines Based on Novel Hydrazine Labeling Reagents

Modified cytosines are important epigenetic marks that exert critical influences in a variety of cellular processes in living organisms. However, biological functions of rare modified cytosines, especially certain functions of 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), are still unclear due to the extremely low abundance in biological samples. In this work, a series of novel hydrazine-based reagents, which held a hydrazine group as the reaction group, a hydrophobic triazine group, and two easily charged tertiary amine groups with different alkyl chains for adjusting the hydrophobicity of the labeling reagents, were first explored to label rare modified cytosines such as 5fC and 5caC. The derivatization reaction between 5fC and the labeling reagents was extremely fast, and more than 99% derivatization efficiency could be achieved only by vortexing without additional reaction time. The detection sensitivity of 5fC increased with the increase of the hydrophobicity of the labeling reagents, the best of which was dramatically enhanced by 125-fold. The limit of detection was as low as 10 amol, realizing the most sensitive genome-wide overall quantification for 5fC. Moreover, the labeling reagents were also successfully applied for the detection of 5caC with 100-fold improvement of sensitivity. With this method, we achieved the simultaneous detection of 5fC and 5caC in different mammalian tissues using only about 600 ng of genomic DNA, which was less than one-tenth of the sample consumption for other reported methods, providing an opportunity to monitor 5fC and 5caC in precious samples and biology processes that could not be investigated before.