Active DNA demethylation process critically relies on the intrinsic properties of ten-eleven translocation proteins (Tets), particularly the flanking sequence preference. Challenged by the fact that the proximate bases to the 5-methylcytosine (5mC) are multitudinous and their influence on the Tets/DNA interplay is minute, the current methodologies are very limited in terms of cost, sensitivity and efficiency. Herein, we propose a polymerization retardation isothermal amplification (PRIA) strategy that enables sensitive and fast study of the flanking sequence preference of Tet. By arranging DNA polymerase to repetitively pass DNA strands through an isothermal replication-scission amplification reaction, the tiny difference in the Tet/DNA interplay can be consecutively accumulated and amplified. Low amount sample (80 ng) even multiple samples can be simultaneously analyzed within 10 h on an easily accessible laboratory real-time quantitative PCR instrument. For a proof-of-concept study, the binding preference (PB) of Tet2 for XmCGX, (X = C, G, T, A) was analyzed by PRIA and computational analysis, showing an order of AmCGT > TmCGA ≈ GmCGC > CmCGG. Furthermore, the binding and oxidation preference in Tet/DNA interplay process was individually considered. By comparative evaluation of the total flanking sequence preference (PT) and the PB, for the first time, we revealed that Tet2 has a lower oxidation preference (PO) to proximal flanking bases and the main contributor to PT of Tet2 is PB. The PRIA strategy, due to its reliable, cost-effective, high efficiency and low sample input, would hopefully advance epigenetic researches and other relative studies.
Keywords: DNA demethylation; Flanking sequence preference; High sensitivity; Polymerization retardation isothermal amplification; Ten-eleven translocation 2 protein.
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