Most m6A modification occurs in m6A motifs having a core ACA sequence, collectively referred to as m6ACA sites. For reliable collection of m6ACA sites, we have established a pipeline to discover all m6ACA sites that are quantifiable by array probes. Most m6ACA sites are consist of a single-ACA that can be profiled for m6A methylation at single-nucleotide resolution. Additionally, m6ACA sites having multiple ACAs or clustered ACAs are profiled jointly at high resolution.
Collection of quantifiable Single-m6ACA Sites
- ACA sites without another ACA present within 40 nt
Fig. 1. Collection pipeline for Single-m6ACA sites.
Step 1: An ACA site with the closest neighboring ACA at least 40 nt away is defined as a quantifiable single-ACA site. An m6ACA site is interrogated by hybridization with an array probe across the (ACA) sequence in its middle. If the site is unmethylated, MazF will cleave the ACA to prevent or greatly reduce the probe binding, thus providing a way to quantify the methylation level. However, if there is another ACA site too close by, it can interfere with, or even prevent, the accurate detection and quantification of the interrogated site. On the other hand, if the distance is greater than 40 nt away from the interrogated site, the neighboring AAC site does not affect the probe signal. Thus, m6A modification in single ACA sites can be quantitatively profiled at single-nucleotide resolution. We collect all quantifiable single ACA sites based on the transcript sequences in the latest Refseq Database.
Step 2: Not all ACA sequences in the RNAs are modified by m6A. All quantifiable single ACA sites collected from Step 1 are then mapped to the actual m6A sites cataloged in the miCLIP dataset[1-4] or the m6A consensus motif near the m6A-seq peak summits, which respectively defines the ultra-high and the high confidence Single-m6ACA sets. These Single-m6ACA sites are finally collected in Arraystar m6A Single Nucleotide Microarrays.
Collection of quantifiable Poly-m6ACA Regions
- Multiple (ACA) sites closely located within a 20-nt region.
Fig. 2. Collection pipeline for Poly-m6ACA sites.
Step 1: If an RNA sequence region contains multiple (ACA) sites within 20-nt, it is defined as a quantifiable poly-ACA region. Instead of resolving m6A modification in each one of the (ACA)s, Poly-(ACA)s are jointly detected and quantified together by an array probe covering the region. That is, if any one of the (ACA)s covered by a probe is unmethylated and cleaved by MazF, the signal from the probe is impacted. So a positive probe signal indicates all the (ACA)s are methylated. Poly-ACA sites detected by microarray are not at single-nucleotide resolution, but very high within 20 nt. A positive signal indicates all individual sites within the Poly-ACA are modified. We collect all these quantifiable poly-ACA regions.
Step 2: Not all ACA sequences in the RNAs are modified by m6A. All quantifiable Poly-ACA regions collected from Step 1 are mapped to the actual m6A sites cataloged in the miCLIP dataset  or the m6A consensus motif near the m6A-seq peak summits, which respectively defines the ultra-high and the high confidence Poly-m6ACA sets. These quantifiable Poly-m6ACA regions are finally collected in Arraystar m6A Single Nucleotide Microarrays.
Analysis of Cluster-m6ACA Regions
When multiple Single-(ACA) or Poly-(ACA) are contained within a 500 nt region and the distance between them is < 100nt, they are merged as one Clustered-ACA region (Fig 3), which can be analyzed by integrating the signals from the individual probes of constituent Single- and Poly-ACA sites. All positive signals from all the probes indicate all the individual m6ACA sites in the cluster are methylated.
m6A Single Nucleotide Array Service
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