A Novel Method On Dna Methylation Modification Based On AGO1 Fusion Methyltransferase

Gene expression regulation is one of the promising techniques to cure disease and genetic disorders.Most of the existing gene regulation technique is irreversible but DNA methylation is a reversible one.It is very important to have a stable carrier for loading methyltransferase into nuclease for successful DNA methylation.In this study,we investigate the novel AGO1 fusion with DNMT3a3 l methyltransferase and its loading into the nucleus and thereby methylation of genes.We identified the unmethylated target gene and developed AGO1 based methyltransferase fused protein and studied its effect in gene suppression by target DNA methylation.The main result of the study are as following;Firstly,we constructed a fused protein of AGO1 and methyltransferase(DNMT3a3l).The two protein was fused with the help of linker(5*G3S).Online simulation of the fusion protein was done and prepared the fused protein of two binding patterns.After that two fusion pattern was designed and observed the loading using confocal microscopy,where DNMT3a3l-linkerAGO1 shown better result inside the nucleus based on the fluorescence contrast.Then we also monitor the expression of the fusion and did transfection of fused protein with SiRNA and observed the loading efficiency,which shows the loading of both fused protein and SiRNA inside the nucleus.Finally,we select cancer-related gene i.e.RASSF1 A gene,and detected the methylation state of the gene using the T-3 cloning technique.We studied the HeLa cell line,Raji cell line,and C-33 A cell line,we found RASSF1 A from the HeLa cell line is unmethylated whereas Raji and C-33 A are methylated at the promoter region of the gene.The gene RASSF1 A expression pattern has been observed in the HeLa cell line after DNA methylation with the fusion protein.Here we also created a dCas9-DNMT3a3 l plasmid and studied the expression change.RASSF1 A promoter region has targeted where SiRNA was used to locate the target in AGO based system whereas SgRNA was used to locate the target in the dCas9 based system.The localization of fused protein and RNAs are observed with fluorescence microscopy indicating successful transfection.The RT-q PCR result indicates both systems shown the change in gene expression level over this particular unmethylated gene promoter region.We developed AGO1 based methylation compound,which successful expression and loading into the nucleus are observed and also use the system to target the unmethylated gene and suppress the expression.We can also target other genes as well by designing SiRNA accordingly.