Most RNAs in nature undergo post-transcriptional modifications, which provide the chemical basis for the diversity of RNA functions, and more RNA modifications are still being discovered. These modifications include N6-methyladenosine (m⁶A), 5-methylcytosine (m⁵C), and pseudouridines (Ψ), etc. Some of these modifications are highly abundant and conserved in RNA and have essential biological functions. For example, m6 A is involved in the post-transcriptional expression of genes in organisms by regulating the shearing, localization, transport, and stability of mRNAs. Lifeasible can provide the tools to further investigate RNA post-transcriptional modifications such as m⁶A, m⁵C, and Ψand provide technical support to enrich and improve RNA epigenetics.

m⁶A detection

The biological function of m⁶A can be precisely investigated by confirming the distribution of m⁶A in RNA. Since the formation of m⁶A does not interfere with the base complementary pairing principle, and it can pair with U as well as normal A, these characteristics make it difficult to detect m⁶A. We can use isotope labeling and thin film chromatography to detect the distribution of m⁶A in RNA.

• We can detect the distribution of m⁶A by isotope labeling and thin-layer chromatography.
• We use m⁶A antibody-based immunoprecipitation and high-throughput sequencing technology (m⁶A -seq or MeRIP) to detect the distribution and modification data of m⁶A at the transcriptome level.

In addition, we also provide other methods, including site-specific cleavage and radioactive labeling followed by ligation-assisted extraction and thin-layer chromatography (SCARLET), photo-crosslinking-assisted m⁶A sequencing strategy (PA- m⁶A -seq), and m6 A-individual nucleotide-resolution crosslinking (miCLIP).

m⁵C detection

m⁵C is a significant RNA post-transcriptional modification in epigenetics. m⁵C is present in both DNA and RNA, so m⁵C is both a representative DNA modification and a significant RNA modification. The distribution of m⁵C can be confirmed by applying the m⁵C modification site detection technique. Our improved RNA m⁵C single-base resolution high-throughput sequencing technique based on ACT three-base random primers combined with bioinformatic analysis can systematically and comprehensively reveal the mRNA m⁵C distribution pattern.

• Sulfite treatment combined with high-throughput sequencing technology.
• 5-azacytidine-mediated RNA immunoprecipitation (Aza-IP) and miCLIP technologies.

We have used m⁵C RNA immunoprecipitation sequencing (m⁵C -RIP-seq) to detect and analyze transcriptome-wide m⁵C modification sites in Arabidopsis.

Ψ modification detection

Using 2D cellulose thin chromatography and high-resolution mass spectrometry allows for quantitative analysis of Ψ modifications.

• RNase H cut specific sites.
• CMCT and SCARLET localization methods.
• The qPCR-based radioactivity-free labeling assay can localize Ψ modifications on mRNA or lncRNA.

These quantitative and localization methods can accurately detect the content and distribution of Ψ modifications better to study the potential functions of Ψ modifications on RNA.

In addition to the m⁶A, m⁵C, and Ψ modification assays provided by Lifeasible, there are a variety of other chemical modifications of RNAs that play a vital role in RNA function and the growth and developmental processes of organisms. Please feel free to contact us to discuss more RNA epimodification sites.

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