Single-Base Resolution Detection of N⁶-Methyladenosine in RNA by Adenosine Deamination Sequencing

Abstract

N 6 -Methyladenosine (m 6 A) is one of the most abundant and prevalent natural modifications occurring in diverse RNA species. m 6 A plays a wide range of roles in physiological and pathological processes. Revealing the functions of m 6 A relies on the faithful detection of individual m 6 A sites in RNA. However, developing a simple method for the single-base resolution detection of m 6 A is still a challenging task. Herein, we report an adenosine deamination sequencing (AD-seq) technique for the facile detection of m 6 A in RNA at single-base resolution. The ADseq approach capitalizes on the selective deamination of adenosine, but not m 6 A, by the evolved tRNA adenosine deaminase (TadA) variant of TadA8e or the dimer protein of TadA-TadA8e. In AD-seq, adenosine is deaminated by TadA8e or TadA-TadA8e to form inosine, which pairs with cytidine and is read as guanosine in sequencing. m 6 A resists deamination due to the interference of the methyl group at the N6 position of adenosine. Thus, the m 6 A base pairs with thymine and is still read as adenosine in sequencing. The differential readouts from A and m 6 A in sequencing can achieve the single-base resolution detection of m 6 A in RNA. Application of the proposed AD-seq successfully identified individual m 6 A sites in Escherichia coli 23S rRNA. Taken together, the proposed AD-seq allows simple and cost-effective detection of m 6 A at single-base resolution in RNA, which provides a valuable tool to decipher the functions of m 6 A in RNA.

Simultaneous Detection of Adenosine-to-Inosine Editing and N6-Methyladenosine at Identical RNA Sites through Deamination-Assisted Reverse Transcription Stalling

“…The deamination efficiency of TadA–TadA8e on adenosine was calculated to be 98.7%, demonstrating a high level of deamination efficiency. The measured deamination efficiency is comparable to the previously reported 99%, which is higher than the deamination efficiency achieved by TadA8e . In addition, we also conducted PAGE analysis to validate the stalling caused by inosine in the RT reaction before and after deamination by TadA–TadA8e.…”

“…The coding sequence of TadA–TadA8e protein was inserted into the pET49b (+) plasmid between MluI and XhoI restriction enzyme digestion sites to generate the plasmid of pET49b-TadA–TadA8e (Figure S1) according to a previous study . The detailed procedures for the expression and purification of TadA–TadA8e protein can be found in the Supporting Information.…”

“…In the realm of m 6 A analysis, some methods have been developed to map m 6 A modifications across various RNA species. These include the antibody-based approaches of m 6 A-seq, MeRIP-seq, and miCLIP, enzyme-assisted methods of m 6 A-SAC-seq, DART-seq, eTAM-seq, AD-seq, and SELECT, chemical-assisted methods of m 6 A-SEAL-seq, GLORI, and m 6 A-ORL-seq, metabolic labeling methods of m 6 A-label-seq, and fluorescence-based detection. − Despite the development of these sophisticated methods for identifying A-to-I editing and m 6 A separately, there remains a notable gap in the ability to simultaneously detect A-to-I editing and m 6 A at the same RNA sites by one method. This limitation underscores a significant challenge in fully understanding the co-occurrence and interplay of these modifications, as well as their collective impact on RNA function and regulation.…”

AlkB-Facilitated Demethylation Enables Quantitative and Site-Specific Detection of Dual Methylation of Adenosine in RNA

“…N 6 -Methyladenosine (m 6 A) is one of the most extensively studied RNA modifications. , In addition to m 6 A, another modification called N 6 , N 6 -dimethyladenosine (m 6,6 A) has also been identified in RNA, particularly in rRNA. , The presence of m 6,6 A at two adjacent adenosines in eukaryotic 18S rRNA and mitochondrial 12S rRNA has been observed, and its impact on rRNA function is currently under investigation . In humans, m 6,6 A is specifically installed at positions 1850 and 1851 in the 18S rRNA .…”

Isothermal Amplification-Based Detection of Single-Base RNA N6-Methyladenosine

“…The most commonly used m 6 A detection methods are mass spectrometry as well as high-throughput sequencing based on m 6 A antibody enrichment, such as MeRIP-seq and miCLIP-seq, which can provide the approximate localization of m 6 A or single-base recognition of m 6 A but with complex procedure, respectively. − Recently, locus-specific m 6 A detection methods (e.g., SCARLET, SELECT, and AD-seq) have been developed. − However, SCARLET requires radioactive labeling, which is challenging to operate and time-consuming; SELECT based on the features of m 6 A selectively hinders single-base elongation and nick ligation, which is prone to false-positive results. Subsequently, Li et al developed a locus-specific and visible m 6 A detection method, m 6 A-Rol-LAMP, which can qualify and quantify single-base m 6 A targets .…”