New breakthrough of alkbh2 detection: real time detection of alkbh2 activity by fluorescence probe

DNA is one of the most important macromolecules in organism However, a variety of internal and external factors, including radiation, may cause DNA alkylation, which is called DNA alkylation damage by scholars Alkylated DNA will cause mismatch in the process of replication, which will lead to various diseases Fortunately, the alkylation of DNA is not irreversible There is a kind of DNA repair enzyme in human body, which can repair damaged DNA into normal DNA Among many DNA repair enzymes, alkbh2 is the only known enzyme that can demethyl 1-methyladenine (M1 a) and 3-methylcytosine (M3 C) in double stranded DNA Although alkbh2 can repair DNA effectively, it will lead to many adverse consequences if it exists too much in organism At present, the determination methods of ALKBH2 activity in vitro include LC-MS, PAGE and so on These methods have their own advantages, but they can not be continuously measured and are lack of selectivity for ALKBH2 Figure 1 Working mechanism of alkbh2 probe (source: angelw Chem Int ed.) Recently, Professor Eric T Kool, Department of chemistry, Stanford University, USA, reported a fast and real-time method to detect the activity of alkbh2 In this method, the activity of alkbh2 in vitro and cell lysate was determined directly by using a fluorescent probe which was responsive to alkbh2 In the probe designed by the authors, the electron deficient M1 A is adjacent to the electron rich pyrene In normal state, the probe shows fluorescence quenching state due to photo induced electron transfer effect Once it undergoes methyl removal reaction under the action of demethylase alkbh2, the quenching effect will disappear, and the probe emits bright fluorescence (Fig 1) This achievement was published in German Applied Chemistry (DOI: 10.1002 / anie 201807593) under the title of "fluorescence probes of alkbh2 measure DNA alkylation repair and drug resistance responses" Table 1 Structure and properties of various probes (source: angelw Chem Int ed.) there is more than one enzyme that can catalyze demethylation in vivo, among which the function of alkbh3 and alkbh2 are very similar, so the primary problem facing the author is how to effectively remove the interference of alkbh3 According to the structural characteristics of alkbh3 probes previously reported, the authors designed probes with different orientations (Table 1, 1-4), different lengths (Table 1, 5-7), different emission wavelengths (Table 1, 8-11) and hairpin structures (Table 1, 12-13p) to explore the influence of these factors on probe selectivity By comparing 1 - 4, it is found that only the naturally oriented β - terminal epimer (1) can effectively determine alkbh2, and the relative position of pyrene and m1a is also very important In addition, the increase of length does not seriously affect the performance of the probe, but from the existing data, the shorter the sequence, the more obvious the change of fluorescence intensity of the probe (5 - 7) Although the change of the emission wavelength does not affect the probe's detection of alkbh2, it is strange that probe 8 did not respond to alkbh2, and the author did not explain this abnormal phenomenon After the preliminary structure of the probe was determined, the probe was further modified Because alkbh2 tends to interact with the double chain structure, while alkbh3 prefers the single chain structure, the hairpin structure is introduced into the probe The experimental results also show that the selectivity of the probe 12 with this structure to alkbh2 has nearly doubled The author then replaced thymine in 12 with adenine to obtain a probe 13 with twice the brightness Fig 2 A) semi inhibition concentration of PDCA; b) detection of alkbh2 in cell lysate by probe 13P (source: angelw Chem Int ed.) Then, the author took 13 as the target probe to study the performance of its detection of alkbh2 activity Firstly, the semi inhibitory concentration of pyridine 2,4-dicarboxylic acid (PDCA), a demethylase inhibitor, was 2.43 ± 0.12 μ m (Fig 2a), which was consistent with the value reported in the literature, indicating that probe 13 could not only meet the requirements of bioanalysis, but also realize the continuous and real-time analysis of alkbh2 for the first time In addition, in order to determine the activity of alkbh2 in cells, the probe 13 was further modified to obtain a methoxy protected probe 13P, which was applied to the detection of the activity of alkbh2 in cell lysates Compared with normal cells and cancerous cells, probe 13P showed very low signal in cancerous cells (Fig 2b), while when alkylating agent TMZ was added to cancerous cells, the fluorescence signal of probe 13P increased nearly twice Finally, the authors indicated that the probes 13 and 13P can monitor the activity of alkbh2 in vitro and in cytolysates in real time, which is the first reported method that can directly determine the activity of alkbh2 At the same time, the high selectivity of the probe to alkbh2 can quantitatively reflect the activity level of alkbh2 David L Wilson, Andrew a Beharry, Avinash Srivastava, Timothy R O'Connor, and Eric T Kool