Blocker-dUThiophene poly tailing-based method for assessing methyl transferase activity.

In this report, we present a method for the selective and sensitive detection of methyl transferase activity. The method uses a dsDNA probe that contains C3 spacers and is coupled with dUThioTP-TdT polymerase-based poly-tailing. The short dsDNA probe is designed with C3 spacers at both 3' ends to prevent any type of tailing reaction. However, the probe contains a methyl transferase recognition sequence that can methylate adenosines in the palindromic part of both strands. When a specific DpnI endonuclease is introduced, it selectively cleaves the dsDNA probe such that both strands are methylated, unblocking the probe into two separate dsDNA forms with exposed 3' OH groups. This makes the probe susceptible to tailing in the presence of a TdT tailing polymerase. The unblocked probe is then subjected to fluorescent dUThioTP-based tailing, which produces a strong fluorescent signal that indicates the presence of methyl transferase activity. In the absence of methyl transferase, the probe remains in the blocked state and does not undergo fluorescence. This method has a limit of detection of 0.049 U/mL with good selectivity and the potential for accurate MTase analysis.