What Is 2'-O-Methylation and How to Detect It

What Is The Purpose of 2'-O-Methylation?

Molecular Mechanisms Regulating RNA Stability

2'-O-Methylation plays a crucial role in maintaining RNA stability by enhancing the resistance of the ribose backbone to hydrolytic degradation. In the ribose structure of RNA, the presence of the 2'-hydroxyl group (2'-OH) makes it susceptible to hydrolysis. The addition of a methyl group at the 2'-OH position through 2'-O-methylation results in the formation of a 2'-O-methylated structure. This minor chemical modification significantly alters the electron distribution within the ribose, reducing its affinity for nucleases. Consequently, RNA exhibits increased resistance to hydrolytic cleavage, thereby maintaining its stability.

From the perspective of RNA helical structure stability, 2'-O-methylation assists in optimizing intramolecular interactions within RNA molecules. The introduction of methyl groups increases steric hindrance, prompting RNA molecules to adopt more stable conformations and enhancing base-pairing stability. This leads to an overall improvement in the stability of the helical structure.

In the field of virology, viruses have adeptly utilized 2'-O-methylation to achieve immune evasion. For example, the RNA genomes of HIV and SARS-CoV-2 exhibit highly concentrated 2'-O-methylation modifications in specific regions. Compared to eukaryotic organisms, viral methylation patterns are more focused and specific. This modification enables viral RNA to escape recognition and attack by the host immune system. The host's immune surveillance typically relies on recognizing pathogenic nucleic acids; however, 2'-O-methylation alters the structure and chemical properties of viral RNA. This modification makes it difficult for pattern recognition receptors of host immune cells to identify the viral RNA, thus creating favorable conditions for viral survival and replication within the host.

2.Association of Functional Sites with Biological Processes

Within the complex architecture of ribosomes, 2'-O-methylation of ribosomal RNA (rRNA) finely regulates ribosome conformation. As the critical site of protein synthesis, the stability and accuracy of ribosomal conformation are essential for the efficiency and quality of protein production. 2'-O-Methylation modifications occur in specific regions of rRNA, acting as precise "molecular switches" that alter local rRNA structures and subsequently influence the three-dimensional conformation of the entire ribosome. Research has identified the methyltransferase FTSJ2 as a key player in this process. In studies of lung cancer cell proliferation, abnormally high expression of FTSJ2 is closely associated with rapid cancer cell growth. Inhibition of FTSJ2 activity leads to a decrease in rRNA 2'-O-methylation levels, resulting in conformational changes in the ribosome, impaired protein synthesis, and ultimately suppression of lung cancer cell proliferation. This finding underscores the significant role of FTSJ2 in the proliferation of lung cancer cells.

The Cap1 Nm modification of messenger RNA (mRNA) exhibits a dual mechanism in immune recognition. On one hand, the Cap1 N^m modification in normal cells serves as a "self-identifier," aiding the immune system in distinguishing self from foreign nucleic acids and preventing erroneous immune attacks on endogenous mRNA. On the other hand, during tumorigenesis-such as in hepatocellular carcinoma-cancer cells exploit abnormalities in Cap1 N^m modification to evade immune surveillance. By altering the Cap1 N^m modification patterns of mRNA, cancer cells become less recognizable to immune cells, creating favorable conditions for tumor growth and metastasis. This dual mechanism highlights the complexity and importance of mRNA Cap1 N^m modification in immune recognition and provides new targets and insights for tumor immunotherapy.

2'-O-Methylation Detection Techniques

Classical Biochemical Methods

Resistance to Alkaline/Enzymatic Hydrolysis

2'-O-methylated RNA resists degradation by RNases (e.g., RNase T1/T2) and alkaline hydrolysis compared to unmodified RNA. This property allows selective enrichment of methylated fragments for analysis.

Example: RiboMethSeq detects underrepresentation of cleavage sites near methylation sites via sequencing coverage drops.

Reverse Transcription (RT) Stops

At low dNTP concentrations, RT enzymes stall at 2'-O-methylation sites, producing truncated cDNA. Differential amplification under normal vs. low dNTP conditions identifies methylation sites (e.g., 2'-OMe-Seq).

Periodate Oxidation-Based Methods

Nm-Seq and RibOxi-Seq exploit periodate's inability to oxidize 2'-O-methylated ribose. Oxidation-resistant RNA fragments are ligated to adapters and sequenced, enabling methylation mapping.

High-Throughput Sequencing Approaches

Deep sequencing-based approaches for detection of 2'-O-methylated residues in RNA

Nanopore Sequencing: Directly detects 2'-O-methylation through altered electrical signals during RNA translocation, offering single-molecule resolution and quantitative analysis.

Emerging Technologies

Nm-VAQ: A bioinformatic tool for site-specific quantification of methylation levels from NJU-seq data, achieving validation rates >80%.

H2Opred: Hybrid deep learning model predicting 2'-O-methylation sites using sequence and structural features, reducing reliance on experimental data.

Technical Considerations

Sensitivity: Methods like NJU-seq detect methylation levels as low as 1%.

RNA Types: Applicable to mRNA, rRNA, tRNA, lncRNA, and snRNA.

Sample Input: Protocols vary, but most require microgram-scale RNA (e.g., ≥1 µg for RiboMethSeq).

Limitations

Bias: Enzymatic/chemical fragmentation (e.g., in Nm-Seq) may introduce sequence bias, requiring iterative optimization.

Throughput: Classical methods (e.g., RT stops) are labor-intensive compared to sequencing-based approaches.

Recent innovations like nanopore sequencing and integrative platforms (e.g., NJU-seq + Nm-VAQ) are expanding the scope of 2'-O-methylation studies, enabling dynamic and context-aware analysis in diverse RNA species

Nanopore Direct Sequencing Technology

Nanopore direct sequencing technology has introduced significant advancements in single-molecule real-time detection. Unlike traditional sequencing methods that often require complex operations such as sample amplification, nanopore technology enables direct sequencing of individual nucleic acid molecules without the need for cumbersome amplification steps. This approach substantially reduces detection time and eliminates potential errors introduced during amplification.

In studies involving the HeLa cell line, the NanoNm algorithm has demonstrated remarkable capacity for locating modification sites, successfully identifying over 20,000 Nm sites. This achievement not only underscores the algorithm's high sensitivity and accuracy but also provides a rich data foundation for in-depth exploration of the 2'-O-methylation modification landscape within HeLa cells.

For data visualization, nanopore direct sequencing technology utilizes specialized software to convert raw sequencing data into intuitive graphs and charts. For example, by plotting genomic coordinates on the horizontal axis and the intensity of detected modification signals on the vertical axis, researchers can generate distribution maps of modification sites. This visualization allows for clear observation of the positions and distribution patterns of 2'-O-methylation modifications across the genome.

Quantitative analysis of modification levels typically involves calculating the proportion of signal intensity at modification sites relative to the total sequencing signal intensity. Through statistical analysis of large-scale sequencing data, the relative modification level of each site can be determined, enabling precise quantification of 2'-O-methylation modification degrees. This quantitative approach provides robust means for investigating differences in 2'-O-methylation modification levels among various samples or under different physiological or pathological conditions, facilitating a deeper understanding of the regulatory mechanisms of 2'-O-methylation in biological processes.

The Nm-Nano framework for predicting Nm sites. (Doaa Hassan et al,. 2024)

Nm-REP-Seq Enzyme-Chemistry Integrated Detection

The Nm-REP-Seq enzyme-chemistry integrated detection method innovatively employs a synergistic mechanism combining MgR exoribonuclease enrichment with OED oxidative elimination. MgR exoribonuclease specifically recognizes and binds to RNA regions bearing 2'-O-methylation modifications, enriching these regions akin to a precise "molecular sieve" that isolates target modified fragments from complex RNA mixtures. The subsequent OED oxidative elimination step further processes the enriched products by eliminating non-target modifications through oxidation reactions. This results in purer and more accurate detection signals, significantly enhancing detection specificity and sensitivity.

This synergistic mechanism exhibits unique advantages in detecting low-abundance mRNA methylation in Drosophila. As an important model organism, studying methylation of low-abundance mRNA in Drosophila has been challenging, with traditional methods often failing to detect accurately. Utilizing its innovative enzyme-chemistry integrated strategy, Nm-REP-Seq technology successfully achieves effective detection of low-abundance mRNA methylation in Drosophila, providing a powerful tool for in-depth research into gene expression regulatory mechanisms during Drosophila development.

The Nm-REP-Seq technology offers notable advantages, including rapid detection cycles that significantly reduce researchers' waiting times and improve research efficiency. Additionally, it possesses cross-species detection capabilities, enabling accurate detection in model organisms such as mice and Drosophila, as well as human samples, thereby broadening its application scope. In clinical contexts, this technology aids in deepening our understanding of methylation regulatory mechanisms of disease-associated genes, providing important evidence for early disease diagnosis, disease monitoring, and the development of personalized therapeutic strategies. Consequently, it holds expansive potential for future applications.

Advantages of 2'-O-Methylation Detection

Featured Techniques and Services

2'-O-Methylation Sequencing

Applications of 2'-O-Methylation Detection

1. Disease Diagnosis and Research

In the field of disease diagnosis, tissue-specific Nm sites in liver cancer hold significant value. Studies have revealed that certain genes in liver cancer tissues exhibit unique 2'-O-methylation modification patterns, which differ markedly from those in normal tissues. Detecting these specific Nm sites provides robust evidence for the early diagnosis of liver cancer. Clinical data demonstrate that this detection method achieves a sensitivity of 92.3%, meaning it accurately identifies 92.3% of liver cancer cases, and a specificity of 88.7%, indicating an 88.7% likelihood that a positive result truly reflects the presence of liver cancer. This significantly enhances diagnostic accuracy, enabling timely detection and the formulation of more effective treatment strategies.

In research on the pathogenic mechanisms of congenital muscular dystrophy, 2'-O-methylation detection technology also plays a crucial role. Researchers have identified abnormal 2'-O-methylation levels in disease-related genes in patients. Through extensive analysis of patient samples, the internal links between these abnormal modifications and disease progression have been gradually uncovered. This not only deepens the understanding of the pathogenic mechanisms of congenital muscular dystrophy but also provides a theoretical foundation for developing targeted therapies. The technology allows for precise localization of molecular changes associated with the disease, offering new hope for addressing this challenging condition.

2. Biological Development Research

In the field of biological development, 2'-O-methylation detection technology has been instrumental in constructing temporal methylation maps during zebrafish embryo development. This technology enables researchers to accurately detect methylation modifications at different developmental stages, facilitating the creation of detailed methylation profiles.

Analysis of dynamic changes in rRNA modifications across developmental stages reveals that 2'-O-methylation levels in rRNA are relatively low during early embryonic development and gradually increase as development progresses, peaking at specific stages. These changes are closely associated with critical events such as cell proliferation, differentiation, and organ formation, highlighting the regulatory role of 2'-O-methylation in embryonic development.

For example, during the critical period of neural tube closure, significant changes in rRNA methylation regulation are observed. Enhanced 2'-O-methylation in specific regions may influence ribosome function, thereby regulating the expression of related genes and ensuring proper neural tube closure. This case underscores the value of detection technology in elucidating biological development mechanisms, providing new insights into the processes of embryonic development.

This section highlights the diverse applications of 2'-O-methylation detection in both disease research and biological development, demonstrating its potential to advance understanding and improve outcomes in these fields.

References:

1. Zhou F, Aroua N, Liu Y, Rohde C, Cheng J, Wirth AK, Fijalkowska D, Göllner S, Lotze M, Yun H, Yu X, Pabst C, Sauer T, Oellerich T, Serve H, Röllig C, Bornhäuser M, Thiede C, Baldus C, Frye M, Raffel S, Krijgsveld J, Jeremias I, Beckmann R, Trumpp A, Müller-Tidow C. A Dynamic rRNA Ribomethylome Drives Stemness in Acute Myeloid Leukemia. Cancer Discov. 2023 Feb 6;13(2):332-347. DOI: 10.1158/2159-8290.CD-22-0210. PMID: 36259929; PMCID: PMC9900322.
2. Zhou F, Liu Y, Rohde C, Pauli C, Gerloff D, Köhn M, Misiak D, Bäumer N, Cui C, Göllner S, Oellerich T, Serve H, Garcia-Cuellar MP, Slany R, Maciejewski JP, Przychodzen B, Seliger B, Klein HU, Bartenhagen C, Berdel WE, Dugas M, Taketo MM, Farouq D, Schwartz S, Regev A, Hébert J, Sauvageau G, Pabst C, Hüttelmaier S, Müller-Tidow C. AML1-ETO requires enhanced C/D box snoRNA/RNP formation to induce self-renewal and leukaemia. Nat Cell Biol. 2017 Jul;19(7):844-855. DOI: 10.1038/ncb3563. Epub 2017 Jun 26. PMID: 28650479.
3. Li, H., Huo, Y., He, X. et al. A male germ-cell-specific ribosome controls male fertility. Nature 612, 725–731 (2022). https://doi.org/10.1038/s41586-022-05508-0
4. Parks MM, Kurylo CM, Dass RA, Bojmar L, Lyden D, Vincent CT, Blanchard SC. Variant ribosomal RNA alleles are conserved and exhibit tissue-specific expression. Sci Adv. 2018 Feb 28;4(2):eaao0665. DOI: 10.1126/sciadv.aao0665. PMID: 29503865; PMCID: PMC5829973.
5. Jansson MD, Häfner SJ, Altinel K, Tehler D, Krogh N, Jakobsen E, Andersen JV, Andersen KL, Schoof EM, Ménard P, Nielsen H, Lund AH. Regulation of translation by site-specific ribosomal RNA methylation. Nat Struct Mol Biol. 2021 Nov;28(11):889-899. DOI: 10.1038/s41594-021-00669-4. Epub 2021 Nov 10. PMID: 34759377.
6. Natchiar SK, Myasnikov AG, Kratzat H, Hazemann I, Klaholz BP. Visualization of chemical modifications in the human 80S ribosome structure. Nature. 2017 Nov 23;551(7681):472-477. DOI: 10.1038/nature24482. Epub 2017 Nov 15. PMID: 29143818.
7. Schosserer M, Minois N, Angerer TB, Amring M, Dellago H, Harreither E, Calle-Perez A, Pircher A, Gerstl MP, Pfeifenberger S, Brandl C, Sonntagbauer M, Kriegner A, Linder A, Weinhäusel A, Mohr T, Steiger M, Mattanovich D, Rinnerthaler M, Karl T, Sharma S, Entian KD, Kos M, Breitenbach M, Wilson IB, Polacek N, Grillari-Voglauer R, Breitenbach-Koller L, Grillari J. Methylation of ribosomal RNA by NSUN5 is a conserved mechanism modulating organismal lifespan. Nat Commun. 2015 Jan 30;6:6158. doi: 10.1038/ncomms7158. Erratum in: Nat Commun. 2016 May 11;7:11530. DOI: 10.1038/ncomms7158 PMID: 25635753; PMCID: PMC4317494.
8. Zhang P, Huang J, Zheng W, Chen L, Liu S, Liu A, Ye J, Zhou J, Chen Z, Huang Q, Liu S, Zhou K, Qu L, Li B, Yang J. Single-base resolution mapping of 2'-O-methylation sites by an exoribonuclease-enriched chemical method. Sci China Life Sci. 2023 Apr;66(4):800-818. DOI: 10.1007/s11427-022-2210-0. Epub 2022 Oct 28. PMID: 36323972.
9. Motorin, Y.; Marchand, V. Detection and Analysis of RNA Ribose 2?-O-Methylations: Challenges and Solutions. Genes 2018, 9, 642. https://doi.org/10.3390/genes9120642
10. Sharma, S., Marchand, V., Motorin, Y. et al. Identification of sites of 2'-O-methylation vulnerability in human ribosomal RNAs by systematic mapping. Sci Rep 7, 11490 (2017). https://doi.org/10.1038/s41598-017-09734-9
11. Huang C, Karijolich J, Yu YT. Detection and quantification of RNA 2'-O-methylation and pseudouridylation. Methods (San Diego, Calif.). 2016 Jul 1;103(103). 68-76. PMID: 26853326 [PubMed] PMCID: PMC4921259
12. Wu K., Li Y., Yi Y., et al. (2025). The detection, function, and therapeutic potential of RNA 2'-O-methylation. The Innovation Life 3:100112. https://doi.org/10.59717/j.xinn-life.2024.100112