Precise quantification of N1-Methyladenosine with a site-specific RNase H cleavage-assisted isothermal amplification strategy

Abstract

Specific and accurate determination of N1-Methyladenosine (m1A) is of critical significance to better understand its biological functions. Precise quantification of low level of RNA modification against a high background of homologous normal RNA still remains a big challenge. To solve this issue, a facile site-specific cleavage-assisted isothermal exponential amplification reaction (SSC-EXPAR) is skillfully designed for direct m1A assay. By exploiting the highly selective RNA cleavage, the normal RNA can be disconnected to avoid the false-positive interference, while the intact m1A can mediate and trigger three-way junction structure-based EXPAR (3WJ-EXPAR). The elegant design of m1A-aroused positive signal can guarantee the accurate and reliable detection of low-level m1A. As low as 1 fM of m1A-containing RNA can be accurately determined with a specificity allowing the discrimination of 0.5% m1A against unmodified adenosine (A) at single-base resolution. In virtue of the outstanding sensitivity and specificity, the SSC-EXPAR approach holds great potential in further understanding of m1A-associated biofunctions and the clinical value in diagnosis and therapeutic treatment of human cancers.