N3-methylcytidine (m3C) at position 32 of the anticodon loop is a conserved tRNA modification found in eukaryotic tRNASer, tRNAThr, and tRNAArg. Arraystar m3C HAC-Seq provides accurate and quantitative profiling of the tRNA m3C methylome for epitranscriptomics research.
Hydrazine-aniline cleavage sequencing for the tRNA m3C methylome
HAC-Seq (hydrazine-aniline cleavage sequencing) maps tRNA m3C modifications at single-nucleotide resolution. Isolated tRNAs are treated with hydrazine and aniline (HAC) to cleave the RNA backbone at m3C sites. To confirm specificity, samples are treated with demethylase prior to HAC (DM-HAC) to remove m3C modifications.
Since HAC-generated 5′ fragments contain damaged 3′ ends that preclude adapter ligation, sequencing captures only full-length and 3′ cleaved fragments. Consequently, m3C sites are identified at single-nucleotide resolution by calculating the cleavage ratio.
HAC-Seq (hydrazine-aniline cleavage sequencing) maps tRNA m3C modifications at single-nucleotide resolution. Hydrazine and aniline cleave the RNA backbone at m3C sites; demethylase-treated controls (DM-HAC) confirm specificity, and m3C sites are identified by calculating the cleavage ratio.
Standard m3C HAC-Seq project — custom designs and add-on analyses available on request
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| m3C HAC-Seq Service |
Key advantages of Arraystar m3C HAC-Seq
Provides precise mapping of m3C sites at nucleotide-level accuracy.
Relies on highly specific chemical reactions rather than antibody affinity, eliminating background from non-specific binding and enabling quantitative stoichiometry assessment.
Eliminates false positives by integrating m3C-demethylated samples as controls for significant cleavage induction and rescue.
Enables simultaneous identification of m3C sites and discovery of modification-associated sequence motifs.
Backed by Arraystar's expertise in tRNA research and the broad tRNA technology portfolio.
Detailed bioinformatics with IGV read alignments, sequence motif analysis, and publication-ready graphics.
m3C in tRNA biology, translation, and disease
First identified in 1963, N3-methylcytidine (m3C) at position 32 of the anticodon loop is a conserved tRNA modification found in eukaryotic tRNASer, tRNAThr, and tRNAArg. This modification acts as a critical regulator of translation and cellular physiology.
In mitochondria, METTL8-dependent m3C on tRNAThr/Ser(UCN) is indispensable for protein synthesis, respiratory activity, and neural stem cell maintenance [1]. Cytoplasmic m3C32, mediated by METTL2A/2B/6 on tRNA-Ser-GCT, facilitates efficient AGU codon decoding and drives the translation of cell-cycle and DNA-repair regulators [2].
Beyond translation, m3C plays vital roles in disease and development: DALRD3-dependent modification of tRNA-Arg is crucial for neurological function [3], while METTL6-mediated modification of tRNA-Ser supports pluripotency and tumorigenesis [4]. Furthermore, nuclear METTL8 stabilizes R-loops via its methyltransferase activity, linking tRNA m3C modification machinery to genome organization [5].
From total RNA to single-nucleotide m3C maps
RNA quality and quantity assessment before the project proceeds, with small-RNA-retaining purification.
Isolated tRNAs are treated with hydrazine and aniline (HAC) to cleave the RNA backbone at m3C sites; parallel samples are treated with demethylase prior to HAC (DM-HAC) to remove m3C modifications and confirm specificity.
Because HAC-generated 5′ fragments contain damaged 3′ ends that preclude adapter ligation, sequencing captures only full-length and 3′ cleaved fragments.
High-throughput sequencing of the HAC and DM-HAC libraries.
m3C sites are identified at single-nucleotide resolution by calculating the cleavage ratio, with IGV read alignments and sequence motif analysis.
Detailed bioinformatics analyses included in m3C HAC-Seq
The m3C HAC-Seq provides a wealth of bioinformatics analyses to better understand their biology and facilitate biomarker applications.
m3C tRNA modification research across biology and disease
METTL8-dependent mitochondrial tRNA m3C is indispensable for protein synthesis, respiratory activity, and neural stem cell maintenance (Cell Stem Cell, 2023); DALRD3-dependent tRNA-Arg modification is crucial for neurological function (Nat Commun, 2020).
Cytoplasmic m3C32, mediated by METTL2A/2B/6 on tRNA-Ser-GCT, facilitates efficient AGU codon decoding and drives translation of cell-cycle and DNA-repair regulators (Nat Commun, 2024).
METTL6-mediated modification of tRNA-Ser supports pluripotency and tumorigenesis (Sci Adv, 2020); nuclear METTL8 stabilizes R-loops, linking m3C machinery to genome organization and tumorigenesis (iScience, 2020).
METTL8-dependent m3C on mitochondrial tRNAThr/Ser(UCN) supports respiratory activity and mitochondrial protein synthesis.
Detailed bioinformatics facilitate insights into m3C in tRNA biology, diseases, and biomarker applications.
Official Arraystar sample submission requirements for m3C HAC-Seq
Common questions about m3C HAC-Seq
Key References for m3C HAC-Seq
Arraystar m3C HAC-Seq combines chemical specificity with demethylase-controlled site identification — get a quote and a project timeline tailored to your study.