The Detection Of The Activity And Level Of DNMT1 Based On Self-assembled Probe Signal Amplification Technique

ObjectiveIn this study,magnetic nanoparticles were used as solid-phase carrier to establish the new methods for the detection of DNA methyltransferase-1(DNMT1)expression activity and level by self-assembled nucleic acid probe signal amplification technology.It is hoped that these new methods could be applied to the detection of DNMT1 activity and level in actual serum samples,and provide a new idea for the early diagnosis of tumors.Methods1.Establishment of detecting DNMT1 activity by signal amplification of self-assembled nucleic acid probeFirstly,the DNMT1 action sequence(Biotin-S1'-S2')was fixed on the magnetic beads of streptavidin by the high affinity of streptavidin and biotin.Secondly,the signal amplification probe poly TAMRA was constructed by self-assembly method.The constructed poly TAMRA was connected to Biotin-S1'-S2' by DNA hybridization to generate a significantly amplified fluorescence signal by poly TAMRA containing multiple fluorescent dyes TAMRA.Finally,the target DNMT1 and restriction endonuclease BssHII were added.In the presence of DNMT1,the hemimethylated Biotin-S1'-S2' could be completely methylated,and BssHII could not recognize the completely methylated Biotin-S1'-S2'.thereby keeping DNA from being cut off by BssHII,which retained strong fluorescence signal.In the absence of DNMT1,BssHII can recognize and cut off the hemimethylated Biotin-S1'-S2',causing the weaker fluorescence signal.The experimental conditions such as the amount of streptavidin magnetic beads,Biotin-S1'-S2',S3-TAMRA,poly TAMRA and the binding time of streptavidin magnetic beads were optimized by single factor experiments.Based on this,the method for detecting the concentration dependent activity of DNMT1 was established,and then the methodology evaluation was carried out.2.Establishment of detecting DNMT1 level by signal amplification of self-assembled nucleic acid probeThe DNMT1 monoclonal antibody(McAbDNMT1)was fixed on the carboxyl magnetic beads by EDC/sulfo-NHS activation method to form the immunomagnetic beads.The immunomagnetic beads can capture DNMT1,and then added the DNMT1 polyclonal antibody(PcAbDNMT1)and biotinylated sheep anti rabbit IgG(sheep anti rabbit IgG-Bio)to form the double antibody sandwich immunomagnetic beads..In the presence of the streptavidin,the Biotin-poly FAM was connected to the double antibody sandwich immunomagnetic beads to form a compound.Immunomagnetic beads can capture the target DNMT1 in the sample,and Biotin-poly FAM can realize signal amplification.The dilution ratio of sheep anti rabbit IgG-Bio,the amount of streptavidin and Biotin-poly FAM were optimized,and the detection method of DNMT1 expression level was established,and then the methodology evaluation was carried out.Results1.Establishment of a method for detecting DNMT1 activity by signal amplification of self-assembled nucleic acid probe(1)Characterization of poly TAMRA:agarose gel electrophoresis results showed that poly TAMRA was composed of double-stranded DNA of different lengths,and its amplification-effect was twice that of S3-TAMRA alone.(2)Selection and immobilization of DNMT1 action sequence:experimental results showed that among the four DNMT1-action sequences of Biotin-S1-S2,Biotin-S1-1-S2,Biotin-S1-2-S2 and Biotin-S1'-S2',DNMT1 could play its function well when Biotin-S1'-S2' acted as the substrate of DNMT1,so Biotin-S1'-S2' was selected as DNMT1 action sequence.The average binding efficiency of Biotin-S1'-S2' with streptavidin magnetic beads was 79.37%,indicated that Biotin-S1'-S2' could be successfully fixed to streptavin magnetic beads.(3)Condition optimization:Single factor experiments were carried out to optimize the established method.The optimal condition was 25?L of streptavidin magnetic beads as the optimal addition amount,the optimal concentration of Biotin-S1'-S2' was 300 nmol/L,the optimal concentration of S3-TAMRA was 600 nmol/L,the optimal concentration of poly·TAMRA was 300 nmol/L,and the binding time of streptavidin magnetic beads and biotin was 20 min.(4)Standard curve and evaluation of method:In the linear range from 1 nmol/L to 100 nmol/L,the corresponding standard curve was Y=168.59X+74211.43(X was CDNMT1,Y was fluorescence intensity F),r=0.9863.The limit of detection was 0.5 nmol/L.The relative standard deviation(RSD)was 0.64%-11.20%,and the recoveries in the standard solutions of the low,medium and high concentrations were 78.9%,115.2%and 102.9%,respectively.2.Establishment of a method for detecting DNMT1 level by signal amplification of self-assembled nucleic acid probe(1)Characterization of Biotin-poly FAM:agarose gel electrophoresis showed that Biotin-poly FAM consisted of DNA double strands of different lengths,with the longest strand reaching 500 bp.(2)Characterization of immunomagnetic beads:the result of scanning electron microscopy showed that the surface of immunomagnetic beads was rougher than that of carboxyl magnetic beads,which could preliminarily determined that McAbDNMTi was successfully modified to the surface of carboxyl magnetic beads,and the immunomagnetic beads were successfully constructed.ELISA experiment on their immune activity showed that the immune activity of the immune substance was.not affected.(3)Condition optimization:the single factor method was used to optimize the experimental conditions,and the good results could be obtained at the-concentration of streptavidin 8 ?mol/L,the dilution ratio of sheep-anti rabbit IgG-Bio 1:600,and the concentration of Biotin-poly FAM 700 nmol/L.(4)Standard curve and evaluation of method:In the linear range from 2 nmol/L to 200 nmol/L,the standard curve was Y=0.28814X+0.01782(X was lgCDNMT1,Y is lg(F/F0)),r=0.9962.The limit of detection was 0.05 nmol/L.The relative standard deviation(RSD)was 4.89%?16.27%,and the recoveries in the standard solutions of the low,medium and high concentrations were 140.0%,94.0%and 101.3%,respectively.ConclusionIn this study,magnetic nanoparticles are used as solid-phase carriers,combined with nucleic acid self-assembly technology to establish a new method with low detection limit and strong specificity for the detection of DNMT1 activity and level.This method is expected to be used in the detection of DNMT1 activity and level in actual serum samples and provide reference for clinical research.