Detection of locus-specific N6-methyladenosine modification based on Ag+-assisted ligation and supersandwich signal amplification

Abstract

Emerging evidence reveals that the epitranscriptomic mark N⁶-methyladenosine (m⁶A) plays vital roles in organisms, including gene regulation and disease progression. However, developing sensitive methods to detect m⁶A modification, especially the identification of m⁶A marks at the single-site level, remains a challenge. Therefore, based on target-specific triggered signal amplification, we developed a highly sensitive electrochemical method to detect site-specific m⁶A modifications in DNA. In this work, the m⁶A site in DNA can restrict the ligation assisted by Ag⁺, and this restriction effect can activate the subsequent strand displacement reaction and hybridization chain reaction (HCR), thus achieving signal amplification from the m⁶A site, and finally realizing high sensitivity analysis of m⁶A methylation. Benefiting from the high specificity of base pairs and the extremely weak binding affinity between Ag⁺ and m⁶A, the proposed method was used for not only detecting the target DNA with a putative m⁶A site, but also identifying m⁶A marks at the single-site level in DNA. In addition, this study does not rely on antibodies and radiolabeling, so it has the advantage of cost-effectiveness. Therefore, we believe that the proposed strategy may provide a new perspective for methylation research, which can be used to test more clinical samples in further research.