Journal: bioRxiv

Article Title: Long-range single-molecule mapping of chromatin modification in eukaryotes

doi: 10.1101/2021.07.08.451578

Figure Lengend Snippet: (A)The experiment concept of the BIND&MODIFY. The cells were lightly fixed and permeabilized for the antibody and enzyme passage. The recombinant protein pA-M.EcoGII was located to desired sites under antibody guidance. Then the M.EcoGII activity was locally activated to modify the nearby regions to label the genomic DNA with targeted binding proteins. (B) The upper panel showed the plasmid map of the pA-M.EcoGII. The fused pA-M.EcoGII was cloned into pTXB1 plasmid and purified with compatible IMPACT protein purification system. The lower panel showed the expressed fusion protein structure: Protein A-linker-M.EcoGII-intein-CBD. (C) The Coomassie blue gel stain showed the purity of the purified protein A-M.EcoGII. (D) Methylation of linear lambda DNA by pA-M.EcoGII activates m6A-site dependent DpnI restriction endonuclease digestion. The PCR amplified unmethylated lambda DNA was treated with commercial M.EcoGII, no enzyme, and pA-M.EcoGII. The G A TC m6A methylation dependent restriction endonuclease DpnI digestion showed the comparable methyltransferase activity of the commercial M.EcoGII and our recombinant proteins. DNA marker: 100bp ladder, 100-1510bp(left); 1kb ladder, 250-10,000bp(right). (E) Methylation of linear dsDNA by pA-M.EcoGII inhibits multiple site-specific methylation sensitive restriction endonucleases. The unmethylated DNA template was a 7kb linear dsDNA, which was PCR amplified from pTXB1 plasmid. The DNA template was treated with commercial M.EcoGII, pA-M.EcoGII and no enzyme. These treated DNA templates were each incubated with four restriction endonucleases (BamHI, EcoRV, PciI, PvuII). The BamHI is the m6A methylation insensitive enzyme, and the EcoRV, PciI, PvuII are the m6A methylation sensitive enzyme, with which the digestion could be blocked by corresponding m6A site. Our pA-M.EcoGII recombinant protein showed digestion inhibition on EcoRV, PciI, PvuII digested samples, better than commercial M.EcoGII, as compared to untreated DNA template. DNA marker, 1kb ladder, 250-10,000bp. (F) The antibody affinity assay showed the recombinant pA-M.EcoGII had the affinity to the secondary antibody in two different dilutions (1/120,1/480, 10mg/ml). (G) The experiment outlines of BIND&MODIFY. After light fixation and permeabilization, the cells were tethered to Concanavalin A magnetic beads for the purification in the next steps. Then the cells were incubated with antibody and pA-M.EcoGII with minimal washes. The addition of S-adenosylmethionine to initialize the methylation reaction. The DNA was extracted to prepare the library for ONT nanopore sequencing. After sequencing, the data was processed as genome alignment and m6A base calling.

Article Snippet: Briefly, the cells are 1) lightly fixed and tethered to Concanavalin A (ConA)-coated magnetic beads, 2) incubated with primary antibodies, 3) incubated with pA-M.EcoGII with minimal washes, 4) S-adenosylmethionine is added and incubated at 37°C for 30min to initialize the methylation reaction, and then quenched by 0.1% SDS, 5) extraction of DNA by phenol-chloroform and prepare the library for Oxford Nanopore Technology (ONT) sequencing.

Techniques: Recombinant, Activity Assay, Binding Assay, Plasmid Preparation, Clone Assay, Purification, Protein Purification, Staining, Methylation, Lambda DNA Preparation, Amplification, Marker, Incubation, Inhibition, Magnetic Beads, Nanopore Sequencing, Sequencing