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Single-Base tRNA Modification Sequencing
Single-Nucleotide tRNA Dihydrouridine (D) Modification Seq(CRACI-seq)
CRACI-seq is an antibody-free, base-resolution approach that quantifies dihydrouridine (D) on tRNA. A chemical conversion step captures D as a unique signal while unchanged positions read as-is, yielding base-resolution D quantification.
Arraystar Single-Nucleotide tRNA Dihydrouridine (D) Modification Seq(CRACI-seq) is an end-to-end sample-to-data service for tRNA, from RNA sample QC and tRNA treatment through library construction, sequencing, and bioinformatics. Sites are localized within mature tRNA transcripts with relative coordinates, tRNA gene attribution, anticodon and isotype, and structural-domain localization.
Benefits
In tRNA, dihydrouridine stabilizes loops and local folding, so position-level mapping within the molecule matters. A chemical conversion step records D as a distinct signal while unchanged positions read as-is, resolving each D site at base resolution.
Single-base resolution: pinpoints D sites in tRNA.
Quantitative measurement: the conversion read-out yields reliable modification fractions.
No antibody dependence: eliminates enrichment bias and cross-reactivity concerns.
Functional annotation: Sites are localized within mature tRNA transcripts with relative coordinates, tRNA gene attribution, anticodon and isotype, and structural-domain localization.
Application-ready: supports decoding fidelity, codon use, aminoacylation, and tRNA structural biology across tRNA isotypes.
| Service Name | Price |
|---|---|
| Single-Nucleotide tRNA Dihydrouridine (D) Modification Seq(CRACI-seq) |
Background
In tRNA, the modification stabilizes the folded L-shaped structure and fine-tunes anticodon–codon decoding, so base-resolution mapping within maturation loops and the anticodon is what distinguishes it. A chemically driven conversion selectively modifies dihydrouridine so it is recorded as a distinct reverse-transcription signature, while unmodified positions read normally, mapping D at single-base resolution. This antibody-free readout maps each D site at single-base resolution.
The method was validated for tRNA, sites are localized within mature tRNA transcripts with relative coordinates, tRNA gene attribution, anticodon and isotype, and structural-domain localization. (Ju CW, et al. He C. Nature Communications 2025;16(1):8863., DOI: 10.1038/s41467-025-63918-w).
Key concept: Single-Nucleotide tRNA Dihydrouridine (D) Modification Seq(CRACI-seq) is a base-resolution method in which chemical conversion records D as a distinct signal while unmodified positions read normally, mapping and quantifying D at single-base resolution.
Figure 1. CRACI-seq dihydrouridine detection scheme. A chemical step captures D as a unique signature while unchanged positions stay as-is, giving base-resolution mapping and quantification of dihydrouridine.
Workflow
A streamlined workflow carries the RNA sample to base-resolution dihydrouridine (D) datasets:
Figure 2. Single-Nucleotide tRNA Dihydrouridine (D) Modification Seq workflow.
Bioinformatics
Downstream analysis maps reads to a curated reference, calls CRACI-seq conversion signatures, and reports D stoichiometry per position, adding motif analysis, differential modification, enrichment, and genome-browser tracks.
Deliverables
- Raw D tRNA sequencing FASTQ data files, formatted for GEO/SRA public database submission.
- QC report with BAM alignment files and key mapping metrics.
- High-confidence single-base D tRNA site tables (XLSX) with coordinates, anticodon, isotype, and stoichiometry.
- Single-base D distribution plots across tRNA domains and consensus D motif logos (PDF/PNG).
- Single-base resolution differential D tRNA site tables (XLSX) with fold change and significance.
- Genome browser-compatible D signal track files (bigWig/bedGraph) and a structured tRNA report.
Research Applications
- tRNA decoding fidelity: maps the modification within anticodon and wobble regions.
- Codon use & aminoacylation: relates base-resolution modification to charging and translation.
- tRNA structural biology: localizes sites within D-loop, anticodon, TΨC, and acceptor stem.
- Isotype-resolved analysis: compares modification across tRNA isotypes and isoacceptors.
- Disease-oriented studies: finds differentially modified tRNA sites as biomarkers.
References
[1] Ju CW, Li H, Jiang B, et al. He C. Quantitative CRACI reveals transcriptome-wide distribution of RNA dihydrouridine at base resolution. Nature Communications 2025;16(1):8863. DOI: 10.1038/s41467-025-63918-w.
Sample Requirements
Sample Storage
For cells/tissue: use TRIzol or an RNA-stabilizing reagent, quick-freeze in liquid nitrogen, and keep at –80 °C.
For RNA: dissolve in ethanol or RNase-free water, store at –80 °C, and limit freeze–thaw cycles.
Dispatch Guidelines
Transfer each sample into a 1.5 mL nuclease-free tube.
Close the tube securely with parafilm or a cap lock to preserve integrity.
Send on dry ice with sufficient insulation to sustain 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 20 µg total RNA. |
FAQ
Which tRNA features are annotated?
Sites are localized within mature tRNA transcripts with relative coordinates, tRNA gene attribution, anticodon and isotype, and structural-domain localization. This lets you interpret each site in the context of its host tRNA isotype and structural domain. The method supports both mRNA and tRNA workflows, so you can choose the readout that matches your study.
Does this method measure the modification at single-base resolution?
Yes. A chemically driven conversion selectively modifies dihydrouridine so it is recorded as a distinct reverse-transcription signature, while unmodified positions read normally, mapping D at single-base resolution. This provides single-base resolution and absolute stoichiometry for each site in tRNA.
How does this method differ from antibody-based tRNA profiling?
Antibody-based methods rely on enrichment and offer limited resolution with intrinsic motif bias. This method is antibody-free and delivers base-resolution, quantitative calling in tRNA, avoiding cross-reactivity and enrichment bias. This suits projects needing quantitative site-level mapping rather than region-level enrichment, and it pairs well with orthogonal validation.
How does this service support research on dihydrouridine?
By profileping D at single-base resolution, the service helps researchers explore its roles in RNA structure and stability, providing a strong evidence base for high-impact work.
Why is high-resolution dihydrouridine detection valuable?
Precise D profiling underpins reliable conclusions in RNA biology, making it a valued tool for advancing the field.
!This assay is provided for research applications only and is not intended for clinical diagnosis, treatment, or personal health evaluation.