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Small RNA Modification Sequencing

TRAC Seq — m⁷G — tRNA m⁷G Modification Seq at Single-Nucleotide Resolution

N7-methylguanosine (m⁷G) is a conserved tRNA modification predominantly located at position 46 in the variable loop of certain tRNAs, enzymatically deposited by the METTL1/WDR4 methyltransferase complex. Arraystar m⁷G TRAC-Seq maps m⁷G sites at nucleotide-level accuracy using highly specific chemical reactions, enabling quantitative stoichiometry assessment.

Chemical specificity · single-nucleotide resolution · quantitative stoichiometry

Overview

Chemical-based m⁷G detection without antibody affinity

m⁷G TRAC-Seq (tRNA reduction and cleavage sequencing) is a chemical method for single-nucleotide resolution profiling of m⁷G throughout the tRNA transcriptome. Isolated tRNAs are enzymatically demethylated and treated with NaBH₄/aniline to induce reductive cleavage specifically at m⁷G residues.

The 3′ cleavage fragments are ligated with a sequencing adaptor at their 5′-ends where the precise modification sites are located. The generated cDNA library for each sample is sequenced, and the sequencing data are analyzed for m⁷G sites and modification levels by cleavage scores.

What is m⁷G TRAC-Seq?

TRAC-Seq (tRNA reduction and cleavage sequencing) is a chemical method for single-nucleotide resolution profiling of m⁷G throughout the tRNA transcriptome. It relies on NaBH₄/aniline reductive cleavage at m⁷G residues, with demethylation controls, to identify m⁷G sites and modification levels by cleavage scores.

Service at a Glance

Standard m⁷G TRAC-Seq project — custom designs and add-on analyses available on request

Service NamePrice
m⁷G TRAC-Seq Service

Benefits

Key advantages of Arraystar m⁷G TRAC-Seq

🎯

Single-Nucleotide Resolution

Precise mapping of m⁷G sites at nucleotide-level accuracy.

⚗️

Chemical Specificity

Highly specific chemical reactions, instead of antibody affinity, to eliminate background from non-specific antibody binding and enable quantitative stoichiometry assessment.

🧬

Superior tRNA Coverage

Demethylation step using the Arraystar rtStar™ tRNA Pretreatment Kit to overcome tRNA modification barriers that block reverse transcription, allowing efficient sequencing of heavily modified tRNAs.

📊

Comprehensive Analyses

Simultaneous identification of m⁷G sites and discovery of modification-associated sequence motifs (e.g. RAGGU motif for m⁷G tRNAs).

🔗

Integrated Application

Works together with our tRF&tiRNA sequencing to uncover how m⁷G modification regulates tRF/tiRNA expression levels.

🖼️

Clear, Well-Organized Figures

Detailed bioinformatics analyses with volcano plots, read alignments, and motif analysis for m⁷G in tRNA biology, diseases, and biomarker applications.

Background

m⁷G in tRNA biology and disease

N7-methylguanosine (m⁷G) is a conserved tRNA modification predominantly located at position 46 in the variable loop of certain tRNAs, enzymatically deposited by the METTL1/WDR4 methyltransferase complex. It plays a pivotal role in maintaining tRNA structural integrity through formation of a C13–G22–m⁷G46 base triple, thereby shielding tRNAs from rapid decay pathways.

Functionally, m⁷G is indispensable for stem cell self-renewal and proper lineage commitment during embryonic development [1]. Moreover, m⁷G-modified tRNAs enhance the translation efficiency of codon-enriched mRNAs, particularly those encoding oncoproteins involved in cell cycle progression, proliferation, and growth signaling.

m⁷G dysregulation, especially with METTL1 overexpression, elevates levels of m⁷G-modified tRNAs (e.g. Arg-TCT-4-1, Lys-CTT, Val-AAC), driving tumorigenesis in diverse malignancies including leukemia, glioblastoma, cholangiocarcinoma, and lung cancer [2-4].

Figure 1. m⁷G TRAC-Seq workflow. Isolated tRNAs are enzymatically demethylated and treated with NaBH₄/aniline to induce reductive cleavage specifically at m⁷G residues. The 3′ cleavage fragments are ligated with a sequencing adaptor at their 5′-ends where the precise modification sites are located. The generated cDNA library for each sample is sequenced. The sequencing data are analyzed for m⁷G sites and modification levels by cleavage scores.
Figure 1. m⁷G TRAC-Seq workflow. Isolated tRNAs are enzymatically demethylated and treated with NaBH₄/aniline to induce reductive cleavage specifically at m⁷G residues. The 3′ cleavage fragments are ligated with a sequencing adaptor at their 5′-ends where the precise modification sites are located. The generated cDNA library for each sample is sequenced. The sequencing data are analyzed for m⁷G sites and modification levels by cleavage scores.

Background References

  1. Lin S, et al. Mettl1/Wdr4-Mediated m(7)G tRNA Methylome Is Required for Normal mRNA Translation and Embryonic Stem Cell Self-Renewal and Differentiation. Molecular Cell, 2018. PMID: 29983320
  2. Orellana EA, et al. METTL1-mediated m(7)G modification of Arg-TCT tRNA drives oncogenic transformation. Molecular Cell, 2021. PMID: 34352207
  3. Dai Z, et al. N(7)-Methylguanosine tRNA modification enhances oncogenic mRNA translation and promotes intrahepatic cholangiocarcinoma progression. Molecular Cell, 2021. PMID: 34352206
  4. Ma J, et al. METTL1/WDR4-mediated m(7)G tRNA modifications and m(7)G codon usage promote mRNA translation and lung cancer progression. Molecular Therapy, 2021. PMID: 34371184

m⁷G TRAC-Seq Workflow

From total RNA to single-nucleotide m⁷G maps

1

Sample QC

RNA quality and quantity assessment before the project proceeds, with small-RNA-retaining purification.

2

Demethylation

Isolated tRNAs are enzymatically demethylated to remove modification barriers, using the Arraystar rtStar™ tRNA Pretreatment Kit.

3

Reductive Cleavage & Library Construction

NaBH₄/aniline treatment induces reductive cleavage specifically at m⁷G residues; 3′ cleavage fragments are ligated with a sequencing adaptor at their 5′-ends.

4

High-Throughput Sequencing

The generated cDNA library for each sample is sequenced.

5

Bioinformatics

Sequencing data are analyzed for m⁷G sites and modification levels by cleavage scores, with motif analysis and differential analysis.

Bioinformatics & Deliverables

Detailed bioinformatics analyses included in m⁷G TRAC-Seq

The m⁷G TRAC-Seq includes detailed bioinformatics analyses to facilitate insights into m⁷G in tRNA biology, diseases, and biomarker applications.

Standard Deliverables

Figure 2. Volcano plot for differentially expressed m⁷G-tRNAs.
Figure 2. Volcano plot for differentially expressed m⁷G-tRNAs.
Figure 3. m⁷G TRAC-Seq read alignments for m⁷G site identification.
Figure 3. m⁷G TRAC-Seq read alignments for m⁷G site identification.
Figure 4. Motif analysis of m⁷G site identified by TRAC-Seq.
Figure 4. Motif analysis of m⁷G site identified by TRAC-Seq.

Research Applications

m⁷G tRNA modification research across biology and disease

Stem Cell Biology

m⁷G is indispensable for stem cell self-renewal and proper lineage commitment during embryonic development (Mol Cell, 2018).

Cancer Biology

METTL1 overexpression elevates m⁷G-modified tRNAs (Arg-TCT-4-1, Lys-CTT, Val-AAC), driving tumorigenesis in leukemia, glioblastoma, cholangiocarcinoma, and lung cancer (Mol Cell, 2021; Mol Ther, 2021).

Oncogenic Translation

m⁷G-modified tRNAs enhance translation efficiency of codon-enriched mRNAs encoding oncoproteins involved in cell cycle progression, proliferation, and growth signaling.

tRF/tiRNA Regulation

Combines with tRF&tiRNA sequencing to uncover how m⁷G modification regulates tRF/tiRNA expression levels.

Biomarker Discovery

Detailed bioinformatics support m⁷G studies in tRNA biology, diseases, and biomarker applications.

Sample Requirements

Official Arraystar sample submission requirements for m⁷G TRAC-Seq

RNA Amount & Quality

  • Total RNA input: > 5 µg per sample (official recommended minimum for the entire experiment in a single attempt, including sample QC). Supply twice the recommended minimum to avoid project delays.
  • Purification: TRIzol / RNA precipitation or an RNA isolation kit. Because tRNA is < 200 nt, use a kit specified to retain small RNAs (e.g. Qiagen miRNeasy).
  • Concentration: > 20 ng/µL by Nanodrop; OD260/280 ~2.0 (acceptable 1.7–2.1); OD260/230 > 1.8.
  • Integrity: sharp 18S/28S rRNA bands by gel, or RIN > 7.0 by Bioanalyzer (serum/plasma/exosome/FFPE RNA exempt).
  • DNase treatment: optional for gDNA removal; required if the sample is also used for qPCR.

Shipping Instructions

  • Ship RNA in nuclease-free water (> 20 ng/µL), freeze-dried, or in ethanol; store at −80 °C or in liquid nitrogen.
  • Use nuclease-free certified, screw-cap 1.5 mL microtubes; seal caps with Parafilm; place tubes in a plastic bag.
  • Use 10 kg dry ice as refrigerant; include a signed Project Form and the sample list.
  • Ship to: Arraystar Inc., 9430 Key West Avenue #128, Rockville, MD 20850, USA. Contact us before shipping.

FAQ

Common questions about m⁷G TRAC-Seq

How does TRAC-Seq detect m⁷G at single-nucleotide resolution?
TRAC-Seq (tRNA reduction and cleavage sequencing) is a chemical method for single-nucleotide resolution profiling of m⁷G throughout the tRNA transcriptome. Isolated tRNAs are demethylated and treated with NaBH₄/aniline to cleave specifically at m⁷G residues. 3′ cleavage fragments are ligated with an adaptor at their 5′-ends, where modification sites are located; sites and levels are identified by cleavage scores.
Why use chemical reactions instead of antibodies for m⁷G detection?
The method relies on specific chemical reactions rather than antibody affinity, which reduces background from non-specific binding and enables quantitative stoichiometry assessment. Chemical detection also avoids batch-to-batch variability from enrichment reagents, which matters when modification levels are compared across a sample series rather than simply called present.
Where does m⁷G sit in tRNA and why does position matter?
Within tRNA, m⁷G at position 46 in the variable loop is the best-characterised site, where the modification supports the folded core of the molecule. Because every call carries its relative coordinate and structural domain, a change in the quantified fraction can be read against the structural role of that position.
How is tRNA coverage achieved for heavily modified tRNAs?
A demethylation step using the Arraystar rtStar™ tRNA Pretreatment Kit overcomes modification barriers that block reverse transcription, allowing efficient sequencing of heavily modified tRNAs. This ensures superior tRNA coverage for quantitative m⁷G profiling. The same pretreatment principle underlies Arraystar's optimised tRNA sequencing methodologies.
What bioinformatics analyses are included?
Projects deliver m⁷G sites and modification levels by cleavage scores, volcano plots for differentially expressed m⁷G tRNAs, read alignments for site identification, and motif analysis. Sites are annotated with host tRNA gene, anticodon, isotype and structural domain so that changes can be grouped by tRNA family.
Can m⁷G TRAC-Seq be combined with tRF&tiRNA sequencing?
Yes. m⁷G TRAC-Seq works together with Arraystar tRF&tiRNA sequencing to show how m⁷G modification relates to tRF and tiRNA levels, linking tRNA modification status with the production of tRNA-derived small RNAs. Both services are sample-to-data offerings within Arraystar's tRNA research portfolio.

Selected Publications

Key References for m⁷G TRAC-Seq

  1. Lin S, et al. Mettl1/Wdr4-Mediated m(7)G tRNA Methylome Is Required for Normal mRNA Translation and Embryonic Stem Cell Self-Renewal and Differentiation. Molecular Cell, 2018. PMID: 29983320
  2. Orellana EA, et al. METTL1-mediated m(7)G modification of Arg-TCT tRNA drives oncogenic transformation. Molecular Cell, 2021. PMID: 34352207
  3. Dai Z, et al. N(7)-Methylguanosine tRNA modification enhances oncogenic mRNA translation and promotes intrahepatic cholangiocarcinoma progression. Molecular Cell, 2021. PMID: 34352206
  4. Ma J, et al. METTL1/WDR4-mediated m(7)G tRNA modifications and m(7)G codon usage promote mRNA translation and lung cancer progression. Molecular Therapy, 2021. PMID: 34371184

Ready to Map m⁷G Modifications at Single-Nucleotide Resolution?

Arraystar m⁷G TRAC-Seq combines chemical specificity with quantitative stoichiometry assessment — get a quote and a project timeline tailored to your study.