Simultaneous Detection of Adenosine-to-Inosine Editing and N 6 -Methyladenosine at Identical RNA Sites through Deamination-Assisted Reverse Transcription Stalling.

Adenosine-to-inosine (A-to-I) editing and N 6 -methyladenosine (m 6 A) modifications are pivotal RNA modifications with widespread functional significance in physiological and pathological processes. Although significant effort has been dedicated to developing methodologies for identifying and quantifying these modifications, traditional approaches have often focused on each modification independently, neglecting the potential co-occurrence of A-to-I editing and m 6 A modifications at the same adenosine residues. This limitation has constrained our understanding of the intricate regulatory mechanisms governing RNA function and the interplay between different types of RNA modifications. To address this gap, we introduced an innovative technique called deamination-assisted reverse transcription stalling (DARTS), specifically designed for the simultaneous quantification of A-to-I editing and m 6 A at the same RNA sites. DARTS leverages the selective deamination activity of the engineered TadA-TadA8e protein, which converts adenosine residues to inosine, in combination with the unique property of Bst 2.0 DNA polymerase, which stalls when encountering inosine during reverse transcription. This approach enables the accurate quantification of A-to-I editing, m 6 A, and unmodified adenosine at identical RNA sites. The DARTS method is remarkable for its ability to directly quantify two distinct types of RNA modifications simultaneously, a capability that has remained largely unexplored in the field of RNA biology. By facilitating a comprehensive analysis of the co-occurrence and interaction between A-to-I editing and m 6 A modifications, DARTS opens new avenues for exploring the complex regulatory networks modulated by different RNA modifications.