Specific quantitative detection of N6-methyladenosine at single-base resolution by extension-based isothermal exponential amplification reaction (E-IEXPAR)

Abstract

Background: 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 accurate m⁶A quantification in small mixed samples and cellular RNA specimens.

Significance and novelty: E-IEXPAR streamlines m⁶A detection by eliminating the need for intricate primer design and thermal cycling, which are common in current analytical methods. Its utilization of an extension reaction for the initial identification of m⁶A, coupled with a novel calculation model tailored to E-IEXPAR outcomes, ensures accurate m⁶A selectivity in mixed samples. As a result, E-IEXPAR offers a reliable, straightforward, and potentially economical approach for specifically assaying m⁶A in both biological function studies and clinical research.

Keywords: Bst DNA polymerase; Isothermal exponential amplification reaction; N(6)-methyladenosine; Single-base resolution.