targeted dna methylation using an artificially bisected m.hhai fused to zinc fingers目标是使用人为平分m的dna甲基化。.pdf

Targeted DNA Methylation Using an Artificially Bisected M.HhaI Fused to Zinc Fingers 1 2 2 2 Brian Chaikind , Krishna Praneeth Kilambi , Jeffrey J. Gray , Marc Ostermeier * 1 Chemistry-Biology Interface Graduate Program, Johns Hopkins University, Baltimore, Maryland, United States of America, 2 Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, United States of America Abstract Little is known about the effects of single DNA methylation events on gene transcription. The ability to direct the methylation toward a single unique site within a genome would have broad use as a tool to study the effects of specific epigenetic changes on transcription. A targeted enzyme might also be useful in a therapy for diseases with an epigenetic component or as a means to site-specifically label DNA. Previous studies have sought to target methyltransferase activity by fusing DNA binding proteins to methyltransferases. However, the methyltransferase domain remains active even when the DNA binding protein is unbound, resulting in significant off-target methylation. A better strategy would make methyltransferase activity contingent upon the DNA binding protein’s association with its DNA binding site. We have designed targeted methyltransferases by fusing zinc fingers to the fragments of artificially-bisected, assembly-compromised methyltransferases. The zinc fingers’ binding sites flank the desired target site for methylation. Zinc finger binding localizes the two fragments near each other encouraging their assembly only over the desired site. Through a combination of molecular modeling and experimental optimization in E. co