In this work, the interaction between camptothecin (CPT) analogs and fat mass and obesity associated (FTO) was investigated using spectroscopy and molecular docking. From the experimental results, it was found that the CPT analogs caused the fluorescence quenching of FTO through a static quenching procedure. The binding constants and thermodynamic parameters at three different temperatures, the number of binding sites were obtained, which suggested that the hydrophobic interaction and electrostatic force played major role in the reaction between CPT analogs and FTO. Results revealed that 10-hydroxycamptothecin was the strongest quencher.
X. Cui,Y. Zhou,Y. Zheng,L. Cao,L. Gao,J. Duan,H. Yin,S. Ai
N 6 -methyladenosine (m 6 A) is the most abundant and dynamic reversible modification of mRNA in eukaryotes discovered in the mid-1970s, which is widely involved in various physiological processes. Fat mass and obesity-associated protein (FTO protein) is not only a demethylase for m 6 A, but also a carcinogenic factor for many human cancers. Therefore, it is very important to build a simple and sensitive platform for simultaneous detection of m 6 A and FTO protein. To achieve this aim, a novel photoelectrochemical biosensor was constructed with the antibody-free detection strategy of DTT mediated –SH functionalization of m 6 A triggered by FTO protein catalyzed m 6 A oxidation . To achieve high detection sensitivity, CdS/Bi 2 MoO 6 /MoSe 2 system with matched energy gap was employed as photoactive material. To achieve high detection selectivity , the –CH 3 of m 6 A in the captured RNA sequence (m 6 A RNA) on the electrode surface was oxidized by FTO protein to form hydroxymethyl, followed with the covalent reaction of DTT with hydroxymethyl, making the labeling of m 6 A with –SH. Finally, a CdS nanoparticle was modified on the electrode through the specific covalent reaction between –SH and Cd, achieving the improvement of the photocurrent with the matched energy band . Under the optimal experimental conditions, the biosensor showed linear range from 0.001 to 50 nM and 0.0005–500 μg/L with the detection limit of 0.37 pM and 0.034 ng/L (S/N = 3) for m 6 A RNA and FTO protein, respectively. The applicability of the developed method was assessed by investigating the effect of entacapone on FTO protein activity.
Yang Hongmei,Wang Yafen,Tang Jing,Wang Fang,Chen Zilin
A promising electrochemical strategy for assay of N6-methyladenosine (m6A)/N6-methyladenine (6mA) in RNA/DNA is proposed. The key of this strategy is the end-labeling of nucleic acid, which makes it possible to detect methylation level in unknown sequence. Firstly, the end of m6A-RNA or 6mA-DNA was labeled with sulfhydryl group through T4 polynucleotide kinase (T4 PNK) and then directly assembled on a gold nanoparticle–modified glassy carbon electrode (AuNPs/GCE). Secondly, methylation sites in RNA/DNA were specifically recognized by anti-m6A-antibody, and then, horseradish peroxidase–labeled goat anti-rabbit IgG (HRP-IgG) was further conjugated on the antibody. Thirdly, HRP-IgG catalyzed the hydroquinone oxidation reaction to generate amplified current signal which correlates with the amount of m6A/6mA in nucleic acid. This method showed a wide linear range from 0.0001 to 10 nM for m6A-RNA, 0.001 to 100 nM for 6mA-dsDNA, and 0.0001 to 10 nM for 6mA-ssDNA. The method was successfully applied to detection of m6A/6mA in RNA/DNA from HeLa cells and E. coli cells and validation of the decrease of m6A-RNA in HeLa cells after treatment with FTO protein. Graphical abstract
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