Specific quantitative detection of N < sup > 6 < /sup > -methyladenosine at single-base resolution by extension-based isothermal exponential amplification reaction (E-IEXPAR)

Anal Chim Acta. 2024 May 8;1302:342474. doi: 10.1016/j.aca.2024.342474. Epub 2024 Mar 13.ABSTRACTBACKGROUND: N6-methyladenosine (m6A) is a common modification in RNA, crucial for various cellular functions and associated with human diseases. Quantification of m6A at single-base resolution is of great significance for exploring its biological roles and related disease research. However, existing analysis techniques, such as polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP), face challenges like the requirement for thermal cycling or intricate primer design. Therefore, it is urgent to establish a simple, non-thermal cycling and highly sensitive assay for m6A.RESULTS: Leveraging the inhibitory effect of m6A on primer elongation and uncomplicated feature of the isothermal exponential amplification reaction (IEXPAR), we have developed an extension-based IEXPAR (E-IEXPAR). This approach requires just a single extension primer and one template, simplifying the design process in comparison to the more complex primer requirements of the LAMP methods. The reactions are conducted at constant temperatures, therby elimiating the use of thermal cycling that needed in PCR methods. By combining IEXPAR with an extension reaction, E-IEXPAR can identify m6A in RNA concentrations as low as 4 fM. We have also introduced a new analytical model to process E-IEXPAR results, which can aid to minimize the impact of unmodified adenine (A) on m6A measurements, enabling acc...

Source: Analytica Chimica Acta - April 5, 2024 Category: Chemistry Authors: Fengxia Su Weiliang Liu Kejian Gao Desheng Chen Yongqiang Cheng Zhengping Li Source Type: research