DART-seq Service

Using an engineered fusion protein that specifically binds m6A and creates targeted C-to-U mutations at adjacent sites in fixed cells, DART-seq enables antibody-free, in situ mapping of m6A modifications with single-nucleotide resolution, effectively preserving native cellular RNA architecture. CD BioSciences provides comprehensive DART-seq services, offering researchers a powerful tool to map m6A landscapes with high resolution and cellular specificity.

Basic Principle of DART-seq

DART-seq is an innovative, antibody-free technology designed for high-resolution mapping of N6-methyladenosine (m6A) directly in preserved cellular contexts. The method employs a fusion protein combining the m6A-binding YTH domain with a hyperactive, catalytically dead APOBEC1 deaminase. When expressed in fixed cells, this construct binds to m6A sites and deaminates adjacent unmodified cytosines to uracils, creating permanent C-to-U mutation signatures in the cDNA during reverse transcription. By sequencing the RNA and identifying these mutation patterns, DART-seq allows for transcriptome-wide identification of m6A sites at single-nucleotide precision while preserving spatial and temporal RNA-protein interactions. This approach provides unique insights into m6A distribution and dynamics within intact cellular architectures.

DART-seq primer bead synthesis and RNA primer validation.

Fig.1 DART-seq primer bead synthesis and validation of RNA priming. (Saikia M, et al., 2019)

Features of DART-seq

DART-seq represents a transformative approach in epitranscriptomics by seamlessly merging cellular biology with high-resolution sequencing. This technology uniquely preserves the native subcellular environment while generating precise, nucleotide-level methylation data, enabling researchers to investigate m6A dynamics within authentic physiological contexts rather than from homogenized lysates.

Single-Cell and Spatial Compatibility: Operates in fixed cells, making it compatible with single-cell RNA-seq workflows and potential spatial transcriptomics applications.
Antibody-Free Detection: Eliminates issues related to antibody specificity, batch variability, and epitope accessibility, ensuring more consistent results.
In Situ m6A Mapping: Captures methylation events within the native cellular environment, preserving RNA localization and interaction contexts.
High Signal-to-Noise Ratio: The targeted deamination creates discrete, readable mutation signatures, reducing background noise common in enrichment-based methods.

Our Services

At CD BioSciences, we offer end-to-end DART-seq services to map m6A modifications with unprecedented cellular and nucleotide-level precision. Leveraging optimized construct delivery and fixation protocols, we generate robust mutation signature data that reveals m6A landscapes directly from your cell samples. This service is particularly valuable for studying m6A heterogeneity across cell populations, during dynamic processes like differentiation or infection, and in limited clinical samples where traditional methods are impractical.

Workflow of DART-seq Service

The DART-seq service employs a meticulously controlled, cell-based workflow that transforms living biological samples into precise, nucleotide-resolution m6A maps through engineered deamination and advanced sequencing.

Steps	Description
Cell Culture & DART Construct Delivery	Target cells are cultured under optimal conditions and transfected or transduced with the engineered DART fusion protein construct (e.g., YTH-APOBEC1-CD) to enable intracellular expression of the detection machinery.
Cell Fixation & Permeabilization	Following appropriate incubation, cells are chemically fixed to preserve native RNA-protein interactions and subcellular architecture, then permeabilized to allow access for subsequent enzymatic reactions.
In Situ Deamination Reaction	Fixed cells undergo controlled treatment to activate the catalytically deactivated APOBEC1 domain of the DART construct, initiating targeted deamination of cytosines adjacent to m6A sites, thereby creating stable C-to-U conversion signatures directly in the cellular context.
RNA Extraction & Strand-Specific Library Construction	Total RNA is carefully isolated while preserving mutation signatures. A strand-specific sequencing library is then prepared, incorporating unique molecular identifiers (UMIs) to control for amplification bias and ensure accurate quantification.
High-Throughput Sequencing & Mutation Signature Analysis	Libraries undergo deep sequencing on Illumina platforms. Specialized bioinformatics pipelines align reads, identify C-to-T (cDNA equivalent of C-to-U) conversions, distinguish true DART-seq signals from background noise using statistical modeling, and call high-confidence m6A sites.
Comprehensive Data Integration & Delivery	Clients receive raw and processed data, annotated m6A peak files, genome browser tracks, quality control metrics, and an analytical report correlating m6A landscapes with potential biological functions and cellular states.

Supported Sample Types

Cultured Cells: Adherent and suspension cell lines, primary cells, stem cells, organoids
Fixed Cell Samples: Chemically fixed cell pellets, cytospin preparations, cells on coverslips
Low-Input & Specialized Samples: FACS-sorted cells, rare cell populations, limited clinical specimens

Our Advantages

True In Situ Detection: Preserves cellular and subcellular context of RNA modifications, enabling correlation with spatial organization and local protein interactions.
Single-Nucleotide Precision with Cellular Resolution: Combines base-level accuracy with cell-type specific information, surpassing bulk sequencing limitations.
Antibody-Independent Methodology: Eliminates variability associated with antibody performance, ensuring consistent, reproducible results across experiments.
Compatible with Downstream Multi-Omics Integration: DART-seq data seamlessly integrates with transcriptomic, proteomic, and imaging datasets from the same cellular samples.
Validated for Challenging Sample Types: Optimized protocols enable reliable analysis of primary cells, rare populations, and dynamic biological processes where conventional methods fail.

DART-seq provides a unique bridge between high-resolution m6A mapping and cellular context, offering insights beyond bulk sequencing approaches. For comprehensive epitranscriptomic analysis, CD BioSciences also provides complementary services including miCLIP-seq for biochemical validation, MeRIP-seq for bulk discovery, and LC-MS/MS for absolute quantification, enabling fully customized research strategies. Contact us to discuss how DART-seq can advance your specific research objectives.

Reference

1. Saikia M, Burnham P, Keshavjee S H, et al. Simultaneous multiplexed amplicon sequencing and transcriptome profiling in single cells[J]. Nature methods, 2019, 16(1): 59-62.