Ligation-driven hyperbranched DNA assembly for label-free and sequence-specific measurement of RNA N6-methyladenosine in human breast tissues

Abstract

N6-methyladenosine (m6A) is a critical post-transcriptional regulator, and it is linked to various biological functions (e.g., degradation, export, and translation) and multiple human diseases (e.g., cancers, immune disorders, and neurodegenerative diseases). The current methods for sequence-specific detection of m6A usually suffer from radioactive risk, large sample consumption, washing/separation steps, and false-positive signal. Herein, we develop a ligation-driven hyperbranched DNA assembly-based fluorescent biosensor for label-free and sequence-specific detection of m6A. In this assay, the unmethylated RNA can be identified and cleaved by m6A-sensitive endoribonuclease MazF. The retained intact m6A-RNA is complementary to the padlock probe to form a circular DNA, subsequently initiating ligation-driven hyperbranched rolling circle amplification (HRCA). The obtained HRCA products can be simply quantified with SYBR Gold as a fluorescent indicator. This method can be homogeneously carried out in an isothermal, label-free and antibody-free manner. The limit of detection can reach 0.079 pM. Moreover, this method can differentiate even 0.01 % m6A level in excess coexisting interferences, monitor cellular m6A RNA level with single-cell sensitivity, and differentiate m6A level in breast tumor tissues and normal adjacent tissues, with promising applications in m6A-releated epigenetic research and clinical diagnosis.