RNA N6,2'-O-dimethyladenosine (m6Am) Analysis

Cap-adjacent N6,2'-O-dimethyladenosine (m6Am) is a distinctive RNA modification at the mRNA 5' end that serves as a critical regulator of transcript fate, influencing stability, translation, and immune recognition. CD BioSciences provides specialized, high-fidelity m6Am analysis services, employing validated methods to accurately distinguish m6Am from internal m6A and deliver clear insights into its biological roles.

Overview of N6,2'-O-dimethyladenosine (m6Am)

Cap-adjacent methylation refers to a specific and critical layer of epitranscriptomic regulation that occurs at the very beginning of messenger RNA (mRNA) molecules, precisely at the first transcribed nucleotide next to the 5' cap structure. Among these modifications, N6,2'-O-dimethyladenosine (m6Am) is the predominant and most studied mark when the first nucleotide is an adenosine. m6Am is a unique dual-methylation modification where both the ribose sugar (2'-O-methylation) and the adenine base (N6-methylation) are chemically altered, distinguishing it structurally from the common internal m6A.

Biologically, m6Am plays a pivotal role as a key determinant of mRNA fate: it enhances transcript stability by protecting against decapping and degradation, modulates translation efficiency, and significantly influences the immunogenic properties of RNA—a factor where its presence can help evade innate immune detection. The research significance of m6Am is profound, as its dysregulation is linked to cellular stress responses, viral pathogenesis, and cancer biology, while in applied science, precise engineering of m6Am is essential for optimizing the stability, efficacy, and safety profile of therapeutic mRNAs and vaccines.

Fig.1 Identification of N6,2'-O-dimethyladenosine (m6Am) methyltransferase. (Sun H, et al., 2019)

Our Services

CD BioSciences offers dedicated solutions to overcome the technical challenge of specifically analyzing m6Am amidst the backdrop of abundant internal m6A. We provide a suite of orthogonal techniques, ranging from absolute quantification to transcriptome-wide mapping, that are explicitly designed to isolate the m6Am signal. This empowers your research into cap-dependent regulation with confidence and precision.

m6Am Analysis Solution by CD BioSciences

Our approach combines gold-standard quantitative mass spectrometry with advanced, capture-based sequencing methods, delivering unambiguous data on m6Am abundance and precise mapping of its location at transcription start sites. This empowers researchers to confidently investigate its role in mRNA stability, translation, and immunogenicity.

Workflow of RNA m6Am Analysis Service

To ensure the highest specificity and reliability in m6Am analysis, CD BioSciences follows a rigorous, end-to-end workflow. This standardized process integrates orthogonal validation and expert bioinformatics, guiding your project from consultation to the delivery of publication-ready data.

Initial Consultation & Method Selection

Our project specialists work with you to define precise research objectives and evaluate sample specifics. Based on your goals, we recommend the optimal integrated strategy, typically combining quantitative LC-MS/MS with a specific mapping technique, to deliver both stoichiometric and positional data.

Sample Preparation & Rigorous QC

Upon receipt, total RNA undergoes stringent quality control (bioanalyzer, spectrophotometry) to assess integrity and purity. For mapping services, an additional cap-specific enrichment or selection step is performed to increase the signal-to-noise ratio for 5'-terminal analysis.

Specific m6Am Capture & Library Construction

For sequencing-based mapping, we execute the chosen m6Am-specific protocol (e.g., exonuclease digestion for m6Am-Exo-seq or enzymatic recapping for ReCappable-seq) to isolate target RNA populations. High-quality, strand-specific sequencing libraries are then constructed, preserving 5' end information critical for accurate mapping.

Instrumental Analysis & Data Generation

Prepared libraries are sequenced on Illumina platforms to generate high-depth data. In parallel, an aliquot of RNA is processed for LC-MS/MS analysis to obtain absolute quantification of m6Am, m6A, and other nucleosides, providing an orthogonal validation of modification levels.

Advanced Bioinformatic & Integrated Analysis

Our dedicated pipeline processes sequencing data for precise alignment, stringent peak calling at transcription start sites (TSS), and differential m6Am site analysis. LC-MS/MS data is analyzed for precise nucleoside quantification. Crucially, results from both methods are integrated and cross-validated to generate a high-confidence list of bona fide m6Am modifications and their quantitative changes.

Comprehensive Reporting & Biological Interpretation

You receive a final report containing all raw data, processed results, and publication-quality visualizations (e.g., aggregate plots at TSS, quantification charts). The report includes a clear biological interpretation, correlating m6Am dynamics with potential effects on transcript stability, translation, or relevant pathways in your study system.

Our Advantages

1. Unambiguous Specificity: Achieve precise discrimination between m6Am and internal m6A by integrating quantitative LC-MS/MS with specific enrichment-based sequencing and stringent bioinformatics.
2. Orthogonal Validation: Ensure the highest data reliability through multi-method validation, such as confirming LC-MS/MS results with independent mapping-based techniques.
3. Expertise in Cap Biology: Leverage our specialized knowledge in 5' cap biochemistry and associated technical challenges to guide your project from experimental design through final data interpretation.
4. Therapeutic Development Support: Obtain critical data on how m6Am incorporation affects RNA stability and immunogenicity to directly support the optimization of synthetic mRNA therapeutics.

As a leading provider of epigenomics research services, CD BioSciences offers comprehensive epitranscriptome analysis solutions. In addition to m6Am analysis, we also provide comprehensive services for various substrate analyses, including m6A, m5C, Ψ, ac4C, m7G, m1A, and RNA adenosine-to-inosine (A-to-I) editing analysis. Contact us today to discuss how to customize our services to help your projects succeed.

Reference

1. Sun H, Zhang M, Li K, et al. Cap-specific, terminal N 6-methylation by a mammalian m6Am methyltransferase[J]. Cell research, 2019, 29(1): 80-82.