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

Abstract

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.

Facile Clamp-Assisted Ligation Strategy for Direct Discrimination and Background-Free Quantification of Site-Specific 5-Formylcytosine

“…It is widely known that mass spectrometry (MS) is a powerful tool for quantification of the global level of epigenetic modifications in biological samples. − However, the MS technique can hardly provide the content of site-specific modified bases. In the future, it is believed that the accurate evaluation of the genomic-wide modified base in a site-specific manner in biological samples may be realized by the integration of the proposed strategy, MS and sequencing techniques.…”

Quantification and mapping of DNA modifications

“…We and others previously utilized 2-bromo-1- benzoic anhydride to simultaneously label 5mC, 5hmC, 5fC, and 5caC in DNA, [98][99][100][101] which led up to 313 fold increase of detection sensitivities of DNA modifications. Similarly, some other chemical derivatization strategies were developed to enable the sensitive detection of DNA modifications, such as glycosylation of 5hmC by T4 β-glucosyltransferase, 102 derivatization of 5fC, 5caC and 5fU by Girard's reagents (Girard D, T, and P) [103][104][105] and hydrazine reagents (Me 2 N, Et 2 N, and i-Pr 2 N), 106 and derivatization of 5fC by dansylhydrazide (DNSH). 107 In addition, 8-(diazomethyl)quinoline (8-DMQ) was used to derivatize the phosphate group of NTPs and modified NTPs, which resulted in the discovery of a series of new modifications occurring in NTPs.…”

Sensitive and Simultaneous Determination of Uridine Thiolation and Hydroxylation Modifications in Eukaryotic RNA by Derivatization Coupled with Mass Spectrometry Analysis

“…Nuclear magnetic resonance (NMR) and mass spectrometry (MS) are pioneer platforms that can provide confirmative information for chemicals. Since the modifications in RNA generally exist in very low abundance and MS typically can offer much better detection sensitivity than that by NMR, MS-based detection therefore has been widely employed in the confirmative detection of nucleic acid modifications. − However, the low ionization efficiencies of uridine modifications make the distinct detection of uridine thiolation and hydroxylation modifications beyond the capability of mass spectrometry. Thus, few studies could offer the quantitative data of uridine thiolation and hydroxylation modifications in RNA, which limits the in-depth investigation of their functions.…”