2.1. Global Quantification of Uracil in DNA

The most straightforward way to quantify the overall uracil content of a DNA sample is a liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method [74]. It is based on enzymatic digestion of the DNA to 2′-deoxy-ribonucleosides using DNase I and nuclease P1, followed by a preparative HPLC purification coupled MS/MS identification of deoxyuridine (dUrd) and employs an isotope labelled internal standard. With this approach, and systematically addressing possible technical pitfalls, the uracil content of the murine and the human genome was determined to be ~0.15 and ~0.08 uracil/10⁶ bases, respectively, considerably lower than suggested previously by other MS-based methods [75,76,77,78]. UNG deficiency led to some increment up to ~1.2 and ~0.35 uracil/10⁶ bases, respectively.

Another approach utilizes alkoxyamine-based aldehyde reactive probes (ARPs) to chemically label the aldehyde group in the deoxyribose moiety at AP sites [79]. Biotinylated ARP reagents were used for the detection of oxidative base damages and AP sites on (ELISA-like) dot blot application [80]. The Ung-ARP assay was developed in Bennett’s group, where specific enzymatic removal of the uracil and detection of the resultant AP sites by biotinylated ARP reagent were combined [81]. Further developments led to two alkoxyamine reagents, AA3 and AA6, associated with increased reactivity and functional groups, appropriate to conjugate with a wide variety of biochemical labels by click chemistry [82,83]. These reagents were used in different applications [84,85,86].

As an independent approach, a new U-DNA sensor protein was developed for multiple purposes, including quick one-step semi-quantitative dot blot application where uracil is directly recognized, without any further enzymatic or chemical reactions [28]. For this, the inactive D145N/H268N double mutant of human UNG was used as a starting construct from which the N′-terminal 84 residues were deleted to eliminate undesired protein–protein interaction surfaces (ΔUNG sensor) [87,88]. It was demonstrated that such UNG-based sensor equipped with 3xFLAG tag is an appropriate tool in dot blot application to quantify uracil as compared to a standard with known uracil content [28]. Uracil levels were determined in both bacterial (wt, ung^−/−, and also ung^−/− dut^−/− double mutant E. coli), and higher eukaryotic (Drosophila S2 cells, as well as human colon cancer cell line HCT116) genomes upon treatments with thymidylate biosynthesis inhibitory drugs. The fast and straightforward dot blot applications do not require mass spectrometry infrastructure; however, the mass spectrometric methods provide higher accuracy, especially at low uracil levels.