Single-Nucleotide RNA m7G Modification Seq (AlkAniline-Seq) reads two marks: m7G and m3C are both reported at single-nucleotide resolution. Heat under alkaline conditions removes the labile base and leaves an abasic site; pre-existing 5′-phosphates are removed first, and the 5′-phosphate created at the next residue is the only end that accepts an adapter.
Arraystar runs the whole route as one sample-to-data workflow, from RNA QC and the cleavage chemistry through library construction, sequencing and bioinformatics, and returns per-site tables in which every position carries its host transcript, its RNA class and its structural region.
Benefits
In total RNA, m7G and m3C are read together. Maps on tRNA and rRNA are the reproducible core; positions on mRNA and lncRNA are reported as exploratory.
Single-base resolution: pinpoints m7G and m3C positions across the RNA classes present in the sample.
Quantitative stoichiometry: the fraction of reads starting immediately 3′ of the cut is reported as the site level, so a partially modified site keeps its intermediate value.
Two marks in the same assay: m7G and m3C are read together, so both site lists can be compared directly.
Antibody-free chemistry: the call follows the cleavage chemistry, and total RNA enters the reaction directly, without RNA-class fractionation before the chemistry.
Functional annotation: sites are annotated to the reference transcriptome with genomic coordinates, host gene, RNA class and functional region attribution.
Application-ready: supports translation control, miRNA processing, writer-enzyme studies, and disease-oriented work.
One-stop service: sample QC, cleavage chemistry, library construction, sequencing and analysis inside one project, with professional bioinformatics support.
| Service Name | RNA Class | Price |
|---|---|---|
| Single-Nucleotide RNA m7G Modification Seq (AlkAniline-Seq) | mRNA | |
| Single-Nucleotide RNA m7G Modification Seq (AlkAniline-Seq) | mRNA & lncRNA |
Background
m7G and m3C have one thing in common that the assay exploits: the bond that holds the modified base to its ribose is unstable. m7G carries a positive charge on the ring nitrogen, and m3C breaks down in the same way, so heat under alkaline conditions is enough to detach the base and leave an abasic site where a normal residue would be intact.
AlkAniline-Seq converts that instability into a position. Aniline drives beta-elimination at the abasic site, the backbone is cut, and a 5′-phosphate is left on the residue next to the modified base. Pre-existing 5′-phosphates are removed by dephosphorylation first, so that phosphate is the only end in the reaction that can be ligated to an adapter, so the library is built outwards from the cut. Every read therefore begins immediately 3′ of the modified position. The mark follows from the parental base that used to sit there: guanosine reports m7G, and a cytidine cut is annotated as m3C only at positions known to carry m3C (ho⁵C shares the same labile bond and is not assigned from the parental base alone).
Key concept: Single-Nucleotide RNA m7G Modification Seq (AlkAniline-Seq) is a chemistry-based sequencing method in which the labile base of m7G or m3C is removed under heat and alkaline conditions, aniline cleaves the backbone at the resulting abasic site, and the 5′-phosphate created at the next residue directs adapter ligation, so the cut itself reports the modification position.
Figure 1. AlkAniline-Seq detection scheme. Heat and alkali generate the abasic site at m7G and m3C, aniline cleavage leaves a 5′-phosphate at the next residue, and adapter ligation starting from that phosphate is what turns each cut into a readable position.
Workflow
Five steps take a sample from RNA to a single-base m7G and m3C table:
Sample QC: RNA integrity and purity are checked before the chemistry is started.
Abasic-site formation and dephosphorylation: the RNA is held under heat and alkaline conditions so the labile base leaves its ribose, after which pre-existing 5′-phosphates are removed.
Aniline cleavage and library construction: aniline cuts the chain at every abasic site and the resulting 5′-phosphate ends are ligated to sequencing adapters.
Sequencing: the library is sequenced on an Illumina platform at a depth suited to position-level calling.
Bioinformatics: reads are aligned, cut positions are scored nucleotide by nucleotide, and m7G and m3C sites are annotated and quantified.
Figure 2. AlkAniline-Seq workflow, from sample QC through abasic-site formation, aniline cleavage and sequencing to a single-base m7G and m3C table.
Bioinformatics
Reads are aligned and cut positions are scored at every nucleotide, with guanosine positions reported as m7G and cytidine positions as m3C, so the two marks keep separate site lists. Analysis adds the host transcript, RNA class and structural region of each site, site-level comparison between sample groups and genome-browser visualization.
Deliverables
- Raw FASTQ data files, ready for GEO/SRA public database submission.
- QC report with BAM alignment files and core mapping statistics.
- Single-base m7G site tables (XLSX) with transcript coordinates, host gene and site level.
- Single-base m3C site tables (XLSX) with transcript coordinates, host gene and site level.
- Site distribution figures by RNA class and by structural region (PDF/PNG).
- Differential modification tables (XLSX) with fold change and significance.
- Genome-browser track files (bigWig/bedGraph) and a structured project report.
Research Applications
- Writer and eraser studies: read how a change in METTL1 or WDR4 shifts m7G site levels, and how m3C levels follow METTL6 or METTL2A/2B.
- Translation control: relate site-level change to codon-biased translation and to ribosome occupancy on the corresponding codons.
- Cancer research: compare m7G and m3C site profiles between tumour and normal samples or across tumour subtypes.
- Development and disease models: follow site levels as cells differentiate, and in models of m7G-related disease.
- Structure and stability: place modified positions in their structural domain and test the consequences for folding, stability and recognition.
- Method cross-validation: set the chemical call beside antibody-based or other base-resolution maps generated from the same samples.
References
[1] AlkAniline-Seq: profiling of m7G and m3C RNA modifications at single nucleotide resolution. Angewandte Chemie International Edition 2018; DOI: 10.1002/anie.201810946.
[2] Mapping of m7G, m3C, dihydrouridine (D) and 5-hydroxycytidine (ho5C) RNA modifications by AlkAniline-Seq. Methods in Enzymology 2021; DOI: 10.1016/bs.mie.2021.06.001.
[3] Global and single-nucleotide resolution detection of 7-methylguanosine in RNA. RNA Biology 2024; DOI: 10.1080/15476286.2024.2337493.
Sample Requirements
Storage Guidelines
Cells and Tissues: Preserve in TRIzol or an RNA stabilization solution; snap freeze in liquid nitrogen and store at -80 °C.
RNA: Resuspend in ethanol or RNase-free ultrapure water; store at -80 °C and avoid multiple freeze-thaw cycles.
Shipping Instructions
Place the sample in a 1.5 mL RNase-free microcentrifuge tube.
Seal the tube with parafilm or a cap lock to ensure sample integrity.
Ship the package on dry ice with adequate insulation to maintain the required temperature.
| Sample Type | Notes |
|---|---|
| Whole blood | Use EDTA tubes only, as heparin is not compatible with subsequent analytical procedures. |
| Cultured cells | Submission of cell pellets is preferred to ensure high-quality material for processing. |
| Tissue | Provide fresh or frozen specimens, ensuring that necrotic material is strictly avoided. |
| Total RNA | Maintain an OD 260/280 ratio ≥ 1.8 and RIN ≥ 7 with no visible degradation. Submit at least 10 µg total RNA. |
FAQ
Why read m7G and m3C in the same experiment?
The two marks share the same chemistry and the same cut, so one library can carry both lists. Reading them together is useful whenever a study asks whether an m7G change is matched by an m3C change in the same sample, in the same RNA preparation and under the same growth condition.
What makes the position precise?
The library starts where the chemistry stopped. Aniline cleavage leaves a 5′-phosphate on the residue next to the modified base, adapter ligation begins from that phosphate, and the first sequenced base therefore maps to the nucleotide after the mark, which places the modification itself at a single position.
Is an antibody used at any step?
No. The call depends on how the modified base behaves in the reaction, not on how strongly an antibody binds it, so a binding preference cannot decide which positions enter the site list.
How does it relate to other base-resolution m7G methods?
Methods such as TRAC-seq reach m7G on tRNA by first selecting small RNA and using AlkB demethylation and borohydride reduction before aniline cleavage. AlkAniline-Seq keeps the alkaline/aniline route, works on total RNA without RNA-class fractionation before the chemistry, and reports m3C alongside m7G.
Can other RNA classes be reported from the same run?
Yes. The chemistry is not class-specific, so the submitted total RNA supplies every class at once. Sites on tRNA and rRNA are recovered most completely and form the reproducible core; positions on mRNA and lncRNA are exploratory and depend on the sample, the depth and the method.
What is reported for a site that is only partly modified?
A level rather than a presence call. The pipeline divides the reads starting immediately 3′ of the cut by the coverage of that position, so a site modified on part of the RNA pool returns an intermediate value and stays visible in the comparison between groups.