Highly sensitive quantitation of N⁶-methyladenosine in long noncoding RNA at precise location with gap extension- and...

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.