Construction Of Methyltransferase Activity Sensing Systems And Their Application

DNA methyltransferase?MTase?activity plays an important role in the life activities of organisms and can effectively affect the process of gene transcription,cell proliferation and cell aging.In addition,DNA MTase activity also plays a crucial role in gene transcription,eukaryotic cell development and cell differentiation,and even in human diseases such as cancer.The study of the activity of DNA MTase and its inhibitors plays a significant role in the etiological analysis and diagnosis of cancer.This paper constructed a variety of sensing methods for the detection of DNA MTase activity,specifically carried out the following several studies:1.A surface plasmon resonance?SPR?sensing system was constructed to detect M.Sss I activity in combination with chain cycle and Au NPs amplification techniques.In the experiment,the carefully designed hairpin DNA?HP1?was used as the methylation substrate and the Au NPs were finally modified onto the sensor chip through a series of enzyme digestion reactions.Due to the resonant coupling effect of Au NPs,a strong SPR signal can be generated,thereby achieving response of different SPR signals to different concentrations of M.Sss I.The linear range of response of this method to M.Sss I was 0.5-50 U/m L,and the detection limit was 0.12 U/m L.The sensor has good selectivity to the detection target.Therefore,this SPR sensor will provide a simple,highly sensitive and highly selective platform for detecting M.Sss I activity.2.The colorimetric sensing system for detecting M.Sss I activity was constructed based on the difference in the binding ability of DNA/Au NPs with GO.The experimental results showed that when the concentration of M.Sss I was in the range of 0.2-60 U/m L,the absorbance A value showed a good linear correlation with log C_{M.Sss I},and the detection limit was 0.067 U/m L.Compared with the M.Sss I sensor reported in the literature,this system has no unusually complicated DNA design,avoids expensive molecular markers,and the experimental procedure is relatively simple.Using cheap and readily available experimental instruments,this method is beneficial to large quantities of practical samples.The experimental results show that the sensor system has the advantages of good repeatability,high sensitivity and good selectivity.3.Based on the polymerization and shear coupling reactions,an amplifiedfluorescence sensing system was constructed for sensitive detection of Dam activity.The ingenious point of the experiment is that s1 containing the sequence “GATC”self-hybridizes to form an s1-s1 duplex that specifically recognizes the cleavage by Dam and Dpn I.The product after the cleavage hybridizes with s2 to form a replication template strand,and then amplification reactions occur under the action of the polymerase and the corresponding cleavage enzymes,resulting in a large number of single-stranded b produced.Single-stranded b reacts with hairpin DNA?MB?which modified with luminescent and quenching groups to open the ring and generate fluorescence.In this system,the fluorescence intensity increases as the concentration of Dam increases,and the fluorescence intensity is in good linear correlation with log C_Dam.The linear range of the response of the experimental method to Dam is0.001-100 U/m L,and the minimum detection limit is 3.2×10^-4 U/m L.