tRNA Dihydrouridine CRACI-seq | Single-Base D Mapping on tRNA

Overview

Base-resolution dihydrouridine profiling across tRNA

Dihydrouridine does not extend the aromatic ring system the way most modified bases do, and that is precisely why it rearranges local geometry: it is the mark that keeps tRNA loops flexible. Because it is concentrated in the D-loop and other loop regions, position-level mapping is what reveals its contribution to folding.

Arraystar Single-Nucleotide tRNA Dihydrouridine (D) Modification Seq(CRACI-seq) is an end-to-end sample-to-data service for tRNA, covering RNA sample QC, tRNA treatment, library construction, sequencing and bioinformatics. Sites are localized within mature tRNA transcripts with relative coordinates, gene attribution, anticodon and isotype, and structural-domain context.

What is tRNA CRACI-seq?

CRACI-seq is an antibody-free, base-resolution method for dihydrouridine on tRNA. Chemical conversion records D as a distinct reverse-transcription signature while unmodified positions read normally, so each D site is mapped and quantified at single-base resolution.

Service at a Glance

Standard tRNA dihydrouridine project — custom designs and add-on analyses available on request

Service Name	Price
Single-Nucleotide tRNA Dihydrouridine (D) Modification Seq(CRACI-seq)

Benefits

What CRACI-seq brings to tRNA studies

D resolved position by position

Chemical conversion gives each dihydrouridine its own signature instead of blending loop signal together.

Quantitative D fractions

The conversion read-out at a position yields the modification fraction for that site.

Antibody-free chemistry

No enrichment step means no pull-down bias and no cross-reactivity inside tightly folded tRNA.

Loop and domain context

Sites arrive with structural-domain localization, so D-loop, anticodon and other regions can be compared directly.

Isotype-level view

Calls carry isotype and isoacceptor context, allowing comparisons between tRNA families.

Complete project execution

RNA QC, tRNA treatment, library preparation, sequencing and analysis are delivered together.

Background

Dihydrouridine and the flexibility of tRNA loops

Dihydrouridine is a reduced pyrimidine: the extra saturation on the ring removes part of the base stacking that stabilises rigid helices, which is what makes it a loop-favouring residue. Its distribution across the D-loop and other loop regions of tRNA is therefore structural information, not just a modification count.

CRACI conversion acts on D and converts it into a signature that reverse transcription reports as a discrete event, while unmodified positions read normally. Arraystar applies that chemistry in a tRNA workflow and anchors each call to the mature transcript and its domain (1).

tRNA Dihydrouridine CRACI-Seq Workflow

From total RNA to a base-resolution D profile

A single workflow carries the RNA sample to base-resolution dihydrouridine data: sample QC and tRNA handling, chemical conversion, library construction and sequencing, then tRNA-aware calling and quantification of D positions.

tRNA Dihydrouridine CRACI-Seq Workflow — Figure 2. Single-Nucleotide tRNA Dihydrouridine (D) Modification Seq workflow.

Bioinformatics & Deliverables

Bioinformatics for CRACI-seq

Reads are mapped to a curated tRNA reference, CRACI conversion events are called as dihydrouridine positions, and per-position stoichiometry is reported alongside motif analysis, differential modification, enrichment testing and browser-ready tracks.

Standard Deliverables

Raw sequencing reads (FASTQ) formatted for GEO/SRA deposition.
QC report with sorted BAM files and core mapping statistics.
Single-base D site tables (XLSX) with coordinates, anticodon, isotype and stoichiometry.
D distribution and motif figures across tRNA domains (PDF/PNG).
Differential D tables (XLSX) with fold change and significance.
Genome-browser tracks (bigWig/bedGraph) with a structured tRNA report.

Research Applications

Where tRNA dihydrouridine mapping fits

Loop flexibility and folding

Relates D positions to the loop architecture that governs tRNA folding.

Domain-level comparison

Compares D occupancy between the D-loop, anticodon and other structural domains.

Isotype-resolved analysis

Contrasts D patterns between tRNA isotypes and isoacceptors.

Stress and disease states

Finds differentially dihydrouridylated tRNA positions between conditions.

tRNA stability studies

Tests whether D changes track with tRNA turnover or stability phenotypes.

Sample Requirements

Sample handling and submission for tRNA dihydrouridine

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.

Shipping Instructions

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; heparin is not compatible with subsequent analytical procedures.
Cultured cells	Cell pellets are preferred to ensure high-quality material for processing.
Tissue	Provide fresh or frozen specimens and avoid necrotic material.
Total RNA	Maintain an OD 260/280 ratio ≥ 1.8 and RIN ≥ 7 with no visible degradation. Submit at least 20 µg total RNA.

Submit at least 20 µg total RNA per sample; contact support@arraystar.com if your material falls below the recommended level. Questions: support@arraystar.com

FAQ

Questions we are asked about tRNA dihydrouridine mapping

How is dihydrouridine detected at single-base resolution?

CRACI chemistry converts D into a distinct reverse-transcription signature while unmodified positions read normally. Because the signal sits at the modified nucleotide itself rather than being inferred from enrichment, each D position is located and quantified to a single base inside the host tRNA transcript.

Why is D hard to profile with antibody methods?

D is concentrated in loop regions of a compact, heavily folded molecule, and enrichment-based assays resolve regions rather than individual positions. CRACI-seq is antibody-free, so it interrogates the site directly without depending on antibody access to a structured tRNA target.

Which features are reported with each D site?

Each call carries relative coordinates inside the mature tRNA transcript, host tRNA gene attribution, anticodon and isotype, and structural-domain localization, so the D-loop and other domains can be compared directly rather than pooled into a single modification count for the whole transcript.

How much total RNA is needed?

We ask for at least 20 µg of total RNA per sample. Material should pass QC with an OD260/280 ratio of 1.8 or higher, a RIN of 7 or higher and no visible degradation on the gel. Contact support if your amount falls below the recommended level.

Can D levels be compared between sample groups?

Yes. Differential analysis is part of the standard pipeline, so D stoichiometry at each position can be contrasted between conditions, with fold change and significance reported per site. That makes it possible to follow a specific tRNA position across a treatment series.

Is dihydrouridine profiling useful outside the D-loop?

Sites are annotated by structural domain, so the answer is visible in the data: any D called in the anticodon loop, the TΨC region or elsewhere is reported with that context rather than being pooled with loop signal from the whole transcript.

Selected Publications

Key references for tRNA dihydrouridine

Ju CW, Li H, Jiang B, et al. Quantitative CRACI reveals transcriptome-wide distribution of RNA dihydrouridine at base resolution. Nature Communications, 2025. PMID: 41053065

CRACI-Seq — Single-Nucleotide tRNA Dihydrouridine (D) Modification Seq