Sensitive fluorescent detection of methyltransferase based on thermosensitive poly(N-isopropylacrylamide)
2018
Niu, Longqing | Wang, Xinxin | Mao, Guobin | Li, Zheng | Ji, Xinghu | He, Zhike
DNA methyltransferase (MTase) has a crucial role in many biological processes, its abnormal expression level has been regarded as a predictive cancer biomarker. Herein, a sensitive fluorescence method based on thermosensitive poly (N-isopr-opylacrylamide) was developed to assay of M.SssI activity. When the M.SssI was introduced, dsDNA was methylated at palindromic sequence 5′-CᵐCGG-3′ and became resistant to cleavage by the endonuclease HpaII. Therefore, a biotin modified ssDNA and a FAM modified ssDNA were designed including the recognized sites for both methyltransferase M.SssI and endonuclease HpaII. By SA-biotin intereaction, the DNA was conjugated to thermosensitive poly (N-isopropylacrylamide) modified by SA, the methylated substrate fluorescence was increased with the concentration of M.SssI increasing. The proposed method has a low detection limit of 0.18 U/mL. This simple method can be a useful tool to apply in diagnosis and biomedical research, which was successfully investigated in the serum sample.
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