6533b857fe1ef96bd12b3bff

RESEARCH PRODUCT

Mapping of 7-methylguanosine (m7G), 3-methylcytidine (m3C), dihydrouridine (D) and 5-hydroxycytidine (ho5C) RNA modifications by AlkAniline-Seq

subject

description

Precise and reliable mapping of modified nucleotides in RNA is a challenging task in epitranscriptomics analysis. Only deep sequencing-based methods are able to provide both, a single-nucleotide resolution and sufficient selectivity and sensitivity. A number of protocols employing specific chemical reagents to distinguish modified RNA nucleotides from canonical parental residues have already proven their performance. We developed a deep-sequencing analytical pipeline for simultaneous detection of several modified nucleotides of different nature (methylation, hydroxylation, reduction) in RNA. The AlkAniline-Seq protocol uses intrinsic fragility of the N-glycosidic bond present in certain modified residues (7-methylguanosine (m7G), 3-methylcytidine (m3C), dihydrouridine (D) and 5-hydroxycytidine (ho5C)) to induce cleavage under heat combined with alkaline conditions. The resulting RNA abasic site is decomposed by aniline-driven β-elimination and creates a 5'-phosphate (5'-P) at the adjacent N+1 residue. This 5'-P is the crucial entry point for a highly selective ligation of sequencing adapters during the subsequent Illumina library preparation protocol. AlkAniline-Seq protocol has a very low background, and is both highly sensitive and specific. Applications of AlkAniline-Seq include mapping of m7G, m3C, D, and ho5C in variety of cellular RNAs, including in particular rRNAs and tRNAs.