Electrochemical immunosensor based on hairpin DNA probe for specific detection of N6-methyladenosine RNA

Abstract N6-methyladenosine (m6A) has been identified as the most frequent messenger RNA (mRNA) modification which is the reversible process of RNA methylation modification and related to biological rhythm regulation. In this work, an electrochemical immunosensor was developed for RNA methylation detection via using gold nanoparticles modified Au electrode (AuNPs/Au) as substrate electrode. Firstly, the hairpin DNA probe was assembled on the surface of AuNPs/Au via Au S bond. In the presence of the hairpin DNA, which is the template of assistant DNA and target RNA, the ligase can selectively ligate them as the template of the hairpin DNA to form a long DNA: RNA heteroduplex strand. Successively, anti-N6-methyladenosine antibody (anti-m6A antibody) could be specifically conjugated onto the RNA methylation site, and the horseradish peroxidase labeled goat anti mouse IgG (HRP-IgG) was further conjugated onto the anti-m6A antibody. In the detection buffer solution with H 2 O 2 and hydroquinone, HRP-IgG can catalyze hydroquinone oxidation to generate benzoquinone, resulting in a highly electrochemical reduction signal. This developed immunosensor shows a wide linear range for methylated RNA from 10 fM to 10 nM, low detection limit of 3.35 fM (S/N = 3), high specificity and satisfactory reproducibility. And this immunosensing method owns great potentials for providing the assay of the expression level of methylated RNA in micro-biological system.