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)

Hongru Pian; Jingli Yan; Weiliang Liu; Hui Wang; Zhengping Li

doi:10.1016/j.talanta.2024.127276

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)

Talanta. 2024 Nov 22:284:127276. doi: 10.1016/j.talanta.2024.127276. Online ahead of print.

Authors

Hongru Pian¹, Jingli Yan², Weiliang Liu¹, Hui Wang¹, Zhengping Li³

Affiliations

¹ Beijing Key Laboratory for Bioengineering and Sensing Technology, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, 30 Xueyuan Road, Haidian District, Beijing, 100083, China.
² Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of the Ministry of Education, Key Laboratory of Analytical Science and Technology of Hebei Province (Project Number: 22567620H), College of Chemistry and Materials Science, Hebei University, Baoding, 071002, China. Electronic address: yanjinglichem@hbu.edu.cn.
³ Beijing Key Laboratory for Bioengineering and Sensing Technology, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, 30 Xueyuan Road, Haidian District, Beijing, 100083, China. Electronic address: lzpbd@ustb.edu.cn.

PMID: 39586212
DOI: 10.1016/j.talanta.2024.127276

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.

Keywords: Gap-extension; Ligation; N(6)-methyladenosine; Nucleic acid isothermal amplification.