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

Adenosine-to-inosine (A-to-I) editing and N6-methyladenosine (m6A) 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 m6A 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 m6A 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, m6A, 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 m6A modifications, DARTS opens new avenues for exploring the complex regulatory networks modulated by different RNA modifications.

Single-Nucleotide Resolution Mapping of N6-Methyladenine in Genomic DNA.

N6-Methyladenine (6mA) is a naturally occurring DNA modification in both prokaryotes and eukaryotes. Herein, we developed a deaminase-mediated sequencing (DM-seq) method for genome-wide mapping of 6mA at single-nucleotide resolution. The method capitalizes on the selective deamination of adenine, but not 6mA, in DNA mediated by an evolved adenine deaminase, ABE8e. By employing this method, we achieved genome-wide mapping of 6mA in Escherichia coli and in mammalian mitochondrial DNA (mtDNA) at single-nucleotide resolution. We found that the 6mA sites are mainly located in the GATC motif in the E. coli genome. We also identified 17 6mA sites in mtDNA of HepG2 cells, where all of the 6mA sites are distributed in the heavy strand of mtDNA. We envision that DM-seq will be a valuable tool for uncovering new functions of 6mA in DNA and for exploring its potential roles in mitochondria-related human diseases.

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

N6-Methyladenosine (m6A) is one of the most abundant and prevalent natural modifications occurring in diverse RNA species. m6A plays a wide range of roles in physiological and pathological processes. Revealing the functions of m6A relies on the faithful detection of individual m6A sites in RNA. However, developing a simple method for the single-base resolution detection of m6A is still a challenging task. Herein, we report an adenosine deamination sequencing (AD-seq) technique for the facile detection of m6A in RNA at single-base resolution. The AD-seq approach capitalizes on the selective deamination of adenosine, but not m6A, 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. m6A resists deamination due to the interference of the methyl group at the N6 position of adenosine. Thus, the m6A base pairs with thymine and is still read as adenosine in sequencing. The differential readouts from A and m6A in sequencing can achieve the single-base resolution detection of m6A in RNA. Application of the proposed AD-seq successfully identified individual m6A sites in Escherichia coli 23S rRNA. Taken together, the proposed AD-seq allows simple and cost-effective detection of m6A at single-base resolution in RNA, which provides a valuable tool to decipher the functions of m6A in RNA.

Genome-wide mapping of N 4-methylcytosine at single-base resolution by APOBEC3A-mediated deamination sequencing.

N 4-methylcytosine (4mC) is a natural DNA modification occurring in thermophiles and plays important roles in restriction-modification (R-M) systems in bacterial genomes. However, the precise location and sequence context of 4mC in the whole genome are limited. In this study, we developed an APOBEC3A-mediated deamination sequencing (4mC-AMD-seq) method for genome-wide mapping of 4mC at single-base resolution. In the 4mC-AMD-seq method, cytosine and 5-methylcytosine (5mC) are deaminated by APOBEC3A (A3A) protein to generate uracil and thymine, both of which are read as thymine in sequencing, while 4mC is resistant to deamination and therefore read as cytosine. Thus, the readouts of cytosines from sequencing could manifest the original 4mC sites in genomes. With the 4mC-AMD-seq method, we achieved the genome-wide mapping of 4mC in Deinococcus radiodurans (D. radiodurans). In addition, we confirmed that 4mC, but not 5mC, was the major modification in the D. radiodurans genome. We identified 1586 4mC sites in the genome of D. radiodurans, among which 564 sites were located in the CCGCGG motif. The average methylation levels in the CCGCGG motif and non-CCGCGG sequence were 70.0% and 22.8%, respectively. We envision that the 4mC-AMD-seq method will facilitate the investigation of 4mC functions, including the 4mC-involved R-M systems, in uncharacterized but potentially useful strains.

An enzyme-mediated bioorthogonal labeling method for genome-wide mapping of 5-hydroxymethyluracil.

DNA 5-hydroxymethyluracil (5hmU) is a thymine modification existing in the genomes of various organisms. The post-replicative formation of 5hmU occurs via hydroxylation of thymine by ten-eleven translocation (TET) dioxygenases in mammals and J-binding proteins (JBPs) in protozoans, respectively. In addition, 5hmU can also be generated through oxidation of thymine by reactive oxygen species or deamination of 5hmC by cytidine deaminase. While the biological roles of 5hmU have not yet been fully explored, determining its genomic location will highly assist in elucidating its functions. Herein, we report a novel enzyme-mediated bioorthogonal labeling method for selective enrichment of 5hmU in genomes. 5hmU DNA kinase (5hmUDK) was utilized to selectively install an azide (N3) group or alkynyl group into the hydroxyl moiety of 5hmU followed by incorporation of the biotin linker through click chemistry, which enabled the capture of 5hmU-containing DNA fragments via streptavidin pull-down. The enriched fragments were applied to deep sequencing to determine the genomic distribution of 5hmU. With this established enzyme-mediated bioorthogonal labeling strategy, we achieved the genome-wide mapping of 5hmU in Trypanosoma brucei. The method described here will allow for a better understanding of the functional roles and dynamics of 5hmU in genomes.