Highly sensitive quantitation of N6-methyladenosine in long noncoding RNA at precise location with gap extension- and ligation-based loop-mediated isothermal amplification (GEXL-LAMP)

Abstract

N6-methyladenosine (m6A), a post-transcriptional modification, is abundant in RNAs, plays prominent roles in various biological processes and is closely associated with human health. However, m6A functions remain largely unrevealed due to the lack of highly sensitive and selective techniques to quantify m6A at specific nucleotide positions, as distinguishing m6A from adenosine (A) is challenging. In this work, we have enhanced the selectivity for discriminating A from m6A by up to 265-fold by combining the polymerase selectivity for gap extension with the ligase selectivity for ligation reaction. With the ligation-based loop-mediated isothermal amplification (LAMP), we achieved ultrahigh sensitivity capable of detecting RNA molecules as low as 40 aM. Therefore, the proposed assay allows precise quantification of m6A modifications at one-nucleotide resolution.